The Evolving Landscape of Systemic Sclerosis Pathogenesis: From Foundational Mechanisms to Organ-Specific Modifiers

Abstract

Systemic sclerosis (SSc) is a multifaceted autoimmune disease in which the complex interplay of genetic predisposition and environmental factors triggers aberrant immune responses, ultimately leading to vasculopathy and fibrosis. This review offers a comprehensive overview of current perspectives on SSc pathogenesis, integrating classical concepts with recent breakthroughs enabled by advanced analytical techniques. We delve into the foundational trans-organ pathophysiology of SSc, encompassing epigenetic dysregulation, chronic inflammation, vascular injury, vasculopathy, and fibrosis. Furthermore, we explore the organ-specific modifiers that contribute to the heterogeneity of SSc manifestations across different organ systems, including the skin, gastrointestinal tract, lungs, and heart. Recent studies employing single-cell transcriptomics, spatial proteomics, and epigenomic profiling are highlighted, demonstrating how these technologies are revolutionizing our understanding of SSc cellular and molecular pathology. This evolving landscape of SSc pathogenesis research is critical for identifying novel therapeutic targets and advancing personalized medicine approaches for SSc patients.

1. Introduction

Systemic sclerosis (SSc) is a multisystem connective tissue disorder of unknown origin, defined by three core pathological features: dysregulated immune activity, vascular injury followed by defective neovascularization and vessel remodeling, and the resulting fibrosis of the skin and various internal organs [1]. The etiology of SSc is still unknown, and there is currently no single hypothesis that uniquely explains the variety of pathophysiologic manifestations of the disease. However, our insight into its pathogenesis is rapidly growing, driven by clinical investigations of patient-derived samples, basic science research with animal models, and progress in targeted molecular treatments. Recent breakthroughs in sophisticated analytical methods, particularly single-cell analysis, have revolutionized the field. These advancements are vital for confirming disease mechanisms in patient tissues and for facilitating the discovery of new therapeutic approaches. This manuscript provides a summary of current perspectives on SSc pathogenesis, with a focus on the latest advancements.

2. Systemic Sclerosis Pathogenesis: Foundational Trans-Organ Pathophysiology

2.1. “Genetics” in SSc

Etiological research suggests that SSc results from a combination of genetic and environmental influences. While family history is the leading risk factor [2], concordance for SSc among twins is low, with comparable rates in both monozygotic (4.2%) and dizygotic (5.6%) pairs. One critical finding, however, is that the concordance for possessing autoantibodies is markedly higher in the unaffected monozygotic twin of an SSc patient (95%), compared to a dizygotic twin (60%, p < 0.05) [3]. This observation suggests that genetic factors are associated with autoimmunity, thereby increasing SSc susceptibility, but are insufficient for the development of clinically definite SSc. Consistent with this notion, the majority of SSc susceptibility genes are Human Leukocyte Antigen (HLA) haplotypes and non-HLA genes implicated in immunity and inflammation, which are also implicated in other connective tissue diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE) [4,5]. Beyond influencing susceptibility, genetic factors also determine the severity of SSc. This is demonstrated by multiple case-control and genome-wide association studies showing that variations like single-nucleotide polymorphisms (SNPs) in certain genes correlate with the disease course [6,7,8,9]. For instance, a specific SNP linked to lower levels of interferon regulatory factor 5 (IRF5) is more prevalent among SSc patients with less severe clinical manifestations [10].

2.2. “Epigenetics” in SSc

Epigenetics refers to the mechanisms by which cells regulate and transmit gene expression without alterations to the DNA base sequence. It also encompasses the field of study dedicated to these mechanisms. The pathogenesis of SSc is investigated from three perspectives: immune dysregulation, vasculopathy, and fibrosis. However, the ultimate goal of these investigations is to elucidate the mechanisms through which fibroblasts maintain a persistently activated state across various organs. While fibroblast activation is generally considered a response to the cumulative stimuli from the extracellular microenvironment in vivo, fibroblasts isolated from the skin and lungs of SSc patients retain their activated phenotype even after in vitro serial passage. This observation suggests the existence of a mechanism for maintaining a “pathological memory” intrinsic to these cells. Epigenetics is considered to be one such mechanism. DNA methylation, histone acetylation and methylation, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) have been identified as epigenetic modifications implicated in sustaining the activated state of fibroblasts derived from SSc patients.

Focusing on genetic factors, genome-wide association studies, as noted earlier, have identified that most susceptibility genes for SSc are located within the HLA region and in non-HLA immune-related genes. This suggests that genetically determined immune abnormalities play a central role in the development of SSc [5]. Indeed, antinuclear antibodies are detected in over 90% of SSc patients, and numerous other autoantibodies with pathogenic functions have been identified [11,12]. In the lesional skin of patients with early-stage SSc, perivascular lymphocytic infiltration is observed. This inflammation is thought to induce structural abnormalities in blood vessels characteristic of SSc, such as the loss of capillaries and thickening of small artery walls. Furthermore, it activates fibroblasts, leading to fibrosis. Although the precise mechanisms remain to be fully elucidated, it is proposed that SSc-specific phenotypic changes arise in various cells through epigenetic regulation influenced by environmental factors and are potentially shaped by genetic predisposition. These changes result in the breakdown of immune tolerance, excessive inflammatory responses, impaired vascular remodeling, and persistent and aberrant activation of vascular endothelial cells and fibroblasts. Consequently, these processes contribute to the complex pathogenesis of this disease [13]. Moreover, epigenetic modifications are deeply implicated in the mechanisms by which various cells maintain their pathological phenotypes within the highly fibrotic environment. In summary, epigenetics is considered to be involved in cellular phenotypic changes from two perspectives: the responses to external environmental factors that trigger SSc onset and the mechanisms maintaining pathological homeostasis against the internal microenvironment.

In SSc, the transcription factor FLI1 was the first gene identified as being under epigenetic control of its expression. Wang et al. [14] reported that, in both cultured SSc dermal fibroblasts and lesional SSc skin, CpG methylation is markedly increased in the FLI1 promoter region. Furthermore, in cultured SSc dermal fibroblasts, significant decreases in histone H3 and H4 acetylation have been observed. Immunohistochemical analysis of skin tissue revealed that FLII expression in fibroblasts and vascular endothelial cells is mildly reduced in non-lesional SSc skin compared to healthy skin. A more pronounced reduction was observed in lesional SSc skin [15]. In the skin of Fli1^+/− mice, fibroblasts are constitutively activated and exhibit increased type I collagen production; however, dermal thickening is not histologically apparent. Furthermore, mild structural abnormalities are present in skin microvessels, and phenotypic alterations resembling those in SSc skin vascular endothelial cells are evident at the molecular level [16]. Conversely, when bleomycin-induced SSc model mice are generated using Fli1^+/− mice, an exacerbation of SSc-like phenotypes is observed across all aspects: inflammation, vasculopathy, and fibrosis [17]. These findings indicate that reduced FLI1 expression can induce activation of immune cells, vascular endothelial cells, and fibroblasts, molecularly resembling SSc, but this anomaly alone is insufficient to trigger SSc onset.

KLF5 is a crucial transcription factor regulating diverse fibrotic responses, acting as a pro-fibrotic factor in cardiac fibroblasts and a fibrosis-suppressing factor in renal tubular cells [18,19]. DNA microarray analysis has revealed that the expression of this transcription factor is downregulated in lesional SSc skin [20]. A study using cultured SSc dermal fibroblasts has revealed a marked downregulation of KLF5 expression at both the mRNA and protein levels. It was demonstrated that the application of an epigenetic inhibitor could recover KLF5 expression to levels comparable to those in healthy cells. Moreover, increased CpG methylation in the KLF5 promoter region was also detected [16]. These findings elucidate that KLF5 expression is robustly suppressed by epigenetic mechanisms in SSc dermal fibroblasts.

Building upon these findings, Klf5^+/−; Fli1^+/− mice were generated to serve as a model for skin fibrosis. Notably, these mice not only faithfully recapitulated skin and lung fibrosis pathologies of SSc but also mirrored SSc vasculopathy, inflammation, and autoimmunity. Importantly, inflammation and autoimmunity emerged at 4 weeks of age, vasculopathy at 4–8 weeks, and skin fibrosis at 8–12 weeks. The temporal sequence of these major pathologies mirrored that observed in SSc [16]. Thus, Klf5^+/−; Fli1^+/− mice can be considered a novel SSc model that spontaneously develops the three major SSc pathologies in a manner temporally similar to human SSc. This research highlights a significant finding: by focusing on factors governing pathological memory in SSc patient-derived cells, we can potentially identify key players in the pathogenesis of this disease.

Analyses of DNA methylation, histone modifications, miRNAs, and lncRNAs have been conducted in various cell types derived from SSc patients, including fibroblasts, vascular endothelial cells, CD4⁺ T cells, B cells, and plasmacytoid dendritic cells (pDCs). Consequently, a substantial number of epigenetic aberrations have been reported. Detailed descriptions of individual findings are omitted in this paper, as they are summarized in several review articles; readers are referred to these for further details [21,22,23,24,25,26,27].

2.3. Genetic Polymorphisms of FLI1 and SSc Susceptibility

Yamashita et al. [4] conducted a case-control study in a Japanese population, directly genotyping this FLI1 (GA)n microsatellite. Their findings revealed a significant association between extended repeat alleles of the FLI1 (GA)n microsatellite and increased susceptibility to SSc. Specifically, alleles with 22 or more GA repeats (L alleles) were more frequent in SSc patients compared to healthy controls. Furthermore, these L alleles were associated with reduced FLI1 mRNA levels in healthy individuals, suggesting a functional consequence of this genetic polymorphism on gene expression. This genetic association with FLI1 further strengthens the evidence implicating FLI1 as a key player in SSc pathogenesis, acting not only through epigenetic modifications but also through inherent genetic predispositions that influence its expression and potentially its function. These findings suggest that FLI1 genetic variants, particularly microsatellite polymorphisms, may contribute to the “missing heritability” in SSc.

2.4. Inflammation and Immunological Dysfunction in SSc

As previously described, the interaction between endothelial cells and circulating immune cells, mediated by cell adhesion molecules and chemokines, facilitates the activation of inflammatory cells and their infiltration into SSc-affected organs. Typically, infiltration by T cells, macrophages, and mast cells predominates in SSc-involved skin, whereas B-cell infiltration is comparatively limited [28,29,30,31]. In contrast, numerous lymphoid aggregates, characterized by a substantial accumulation of B cells and relatively fewer T cells and macrophages, are commonly observed in the lung tissue of patients with SSc-associated interstitial lung disease (ILD) [32]. Notably, genes associated with activated B cells are upregulated in SSc-involved skin [20], and the B-cell count in the skin correlates with the progression of skin fibrosis [31]. Consequently, while the composition of infiltrating cell types may vary across different affected organs, increased infiltration of B cells, T cells, and innate immune cells is a shared feature in the organs involved in SSc.

Alterations in T cell subsets are a well-documented feature of SSc. The balance of Th1/Th2 and Th17/Treg immune responses is skewed toward Th2 and Th17 dominance [33,34,35,36], and regulatory T cell (Treg) function is impaired during the active phase of SSc [37]. In the early stage of diffuse cutaneous SSc (dcSSc), serum levels of interleukin-6 (IL-6) and IL-10 are significantly elevated, whereas they decrease to normal levels in the late stage of dcSSc, which is characterized by the regression of skin sclerosis [38]. The cytokine profile in dcSSc displays dynamic changes over time. Initially, IL-4 levels are normal, and they decline as the skin sclerosis resolves. In contrast, serum IL-12 is low in early dcSSc but rises with disease duration, eventually exceeding normal levels in the late stage [33]. The Th17 pathway is also clearly involved; in early dcSSc skin lesions, the expression of IL-17A, IL-21, and IL-22 is increased, though IL-17F is not [36,39]. Systemically, the frequency of circulating Th17 cells and their IL-17 production are elevated, with Th17 counts corresponding to disease activity [34]. Within the skin, the Treg population is also altered, showing an increased proportion of Th2-like Tregs [30].

Currently, the direct role of SSc-related antinuclear antibodies (ANAs), including antibodies against topoisomerase I (topo I), centromere, and RNA polymerase III (RNAP III) antigens, is not fully understood, although a potential role for anti-topoisomerase I antibodies has been suggested (described below). Nevertheless, the strong correlation of these ANAs with clinical manifestations implies that altered B-cell phenotypes may be associated with the core abnormality driving disease progression.

On the other hand, the potential pathogenic role of so-called functional vascular antibodies, particularly those targeting the angiotensin II type 1 receptor (AT1R) and the endothelin-1 type A receptor (ETAR), has been a subject of discussion. However, compelling evidence for their direct contribution to pathophysiology is still lacking, and the clinical utility of testing for these antibodies remains unclear [40,41]. The ultimate proof that an antibody is causative is when passive transfer in an animal model (possibly with a specific genetic background) results in disease features.

Aberrant activation of B cells could occur through genetic and epigenetic mechanisms shared across cell types and/or through complex interactions with other immune and non-immune cells. In SSc, B cells are in a state of continuous activation, demonstrated by increased surface expression of the co-receptor CD19 [42] and the activation markers CD80 and CD86 on memory B cells [43]. This is significant because, in addition to producing antibodies, B cells contribute to pathogenesis through cytokine secretion, antigen presentation, and regulation of macrophages and lymphoid tissue [44]. The critical role of B cells is supported by the efficacy of rituximab, an anti-CD20 antibody that improves skin fibrosis and ILD by depleting B cells, as shown in multiple case series and open-label studies [45,46,47,48]. Furthermore, several case reports and case series have documented the amelioration of calcinosis, digital ulcers (DUs), and arterial stiffness following rituximab therapy [49,50,51]. CD19-targeted Chimeric Antigen Receptor (CAR) T-cell therapy is an emerging investigational treatment for severe and refractory SSc. Initial case studies and small series have demonstrated promising clinical responses, including improvements in skin fibrosis, ILD, and cardiac function, often accompanied by reduced autoantibody levels and disease activity. Supporting data show decreased TGF-β levels in dermal biopsies, improved skin elasticity on elastography, and amelioration of lung and heart fibrosis on imaging and through markers like KL-6. These benefits sometimes allow cessation of other immunosuppressive treatments, with generally manageable safety profiles reported [52,53,54,55]. Thus, B cells contribute to the activation of vascular and fibrotic processes, in addition to immune system activation in SSc, reinforcing the concept that immune cells are upstream mediators in the SSc-specific disease cascade.

Beyond adaptive immune cells, innate immune cells are also abundantly present in SSc-involved organs. In SSc lesional skin, mast cells secrete excessive levels of transforming growth factor-β (TGF-β) [56]. Moreover, M2 macrophages appear to be critical regulators of tissue fibrosis, as the M2 macrophage-associated gene program, which is upregulated in the skin of early SSc patients, is suppressed in conjunction with the resolution of skin fibrosis following treatment with tocilizumab (an anti-IL-6 receptor antibody) [57].

In SSc-affected skin, a specific sequence of events is thought to drive excess IFN-α production by plasmacytoid dendritic cells (pDCs). First, pDCs are recruited to the area around dermal small vessels by elevated levels of the chemoattractant chemerin [58,59]. Concurrently, endothelial cell death provides a source of self-DNA, which interacts with increased local concentrations of LL-37 [60]. This process forms stimulatory self-DNA/LL-37 complexes that are hypothesized to activate pDCs via TLR7 and 9, resulting in high local production of IFN-α.

Furthermore, disease-associated autoantibodies, particularly anti-topoisomerase I antibodies, may contribute to this process. Anti-topoisomerase I antibodies react with nuclear antigens from endothelial cells, and immune complexes formed with nucleic acids, especially RNA, induce IFN-α production from pDCs [61]. The idea that IFN-α contributes to SSc development is supported by clinical and experimental data. A trial using recombinant IFN-α for SSc reported higher withdrawal rates than placebo, with many discontinuing patients showing worsened ILD [62]. Moreover, administering IFN-α for other disorders, like multiple sclerosis or chronic hepatitis C, can trigger SSc or similar symptoms [63,64,65,66,67,68]. This may be explained by a self-amplifying cycle in which continuous IFN-α exposure causes endothelial senescence [69], providing self-DNA that stimulates pDCs to produce more IFN-α, thus driving vascular injury and immune activation. Recently, the discovery of ectopic TLR8 expression on SSc pDCs, a key RNA sensor linked to experimental fibrosis, has added another layer to this mechanism [70]. In addition to their role in IFN-α production, recent findings indicate that pDCs can also contribute to fibrosis through endoplasmic reticulum (ER) stress-mediated mechanisms, as demonstrated by Ferreira et al. [71] who revealed that ER stress induction in pDCs promotes fibroblast activation via direct cell–cell contact, suggesting a novel pathway contributing to fibrosis development in SSc. Collectively, the continuous release of autoantigens from damaged and senescent endothelial cells serves as a fundamental driver of SSc pathology, acting through the induction of chronic inflammation.

2.5. Vascular Injury in SSc

As previously discussed, the pathogenesis of SSc begins with immune dysregulation, while histopathologically detectable structural abnormalities first manifest as vascular damage [72,73,74]. Indeed, vasculopathy is a critical element in the early clinical picture of SSc, manifesting in patient-reported symptoms such as Raynaud’s phenomenon and digital edema [75]. Crucially, the presence of disease-specific autoantibodies, hallmarks of SSc’s autoimmune nature, can be detected even before these initial clinical manifestations emerge, highlighting the early involvement of autoimmunity in vascular injury [75,76,77]. Following this initial vascular insult, the vasculature in SSc undergoes significant structural abnormalities [78]. These changes arise from a combination of dysfunctional vascular remodeling processes and the development of various vascular functional impairments [78].

In the early stages of SSc, capillary fragility leads to capillary dilation, which in turn results in the extravasation of erythrocytes [75]. The observation of dilated and hemorrhaging nailfold capillaries is diagnostically significant, serving as an important early indicator of the disease [76]. As SSc progresses, a progressive loss of capillaries occurs, with capillary numbers gradually diminishing and eventually being replaced by fibrotic tissue [76]. In parallel with this capillary rarefaction and fibrotic replacement, vascular endothelial cells and pericytes, which are crucial components of blood vessels, undergo differentiation into myofibroblasts through processes known as endothelial-to-mesenchymal transition (EndoMT) and pericyte-to-mesenchymal transition (PMT), respectively [78,79]. These transformed cells acquire resistance to apoptosis, or programmed cell death, and exhibit cellular senescence phenotypes. This altered cellular behavior significantly contributes to the establishment and perpetuation of the extensive fibrosis characteristic of SSc [78]. The ensuing loss of capillaries leads to tissue hypoxia, a state of oxygen deficiency, which in turn acts as a potent stimulus for further myofibroblast activation and promotes the fibrotic process across a range of organs, including the skin, lungs, heart, and intestines [78]. In contrast, within arterioles and small to medium-sized arteries, the vascular endothelial cells, when injured, also undergo differentiation into myofibroblasts [78]. Furthermore, the proliferative capacity of vascular smooth muscle cells, another key cell type in blood vessels, and their ability to produce extracellular matrix (ECM) components, are enhanced, culminating in fibrotic stenosis, or narrowing of the blood vessels [78]. The characteristic arterial lesions of SSc arise from an abnormal gathering of myofibroblasts within the vessel wall. This accumulation creates fibroproliferative changes that subsequently impair vascular perfusion [78].

The vascular functional derangements in SSc are multifaceted and encompass a range of abnormalities. These include diminished vascular endothelial function, which refers to the impaired ability of the endothelium to regulate vascular tone and permeability; a reduced capacity for thrombus inhibition, increasing the risk of blood clot formation; impaired physiological anticoagulation mechanisms, further exacerbating the pro-thrombotic state; aberrant expression of cell adhesion molecules and chemokines, which contribute to chronic inflammation and immune cell recruitment to the vessel wall; and an augmented generation of reactive oxygen species (ROS), leading to oxidative stress and cellular damage [17,80,81]. These functional aberrations play a crucial role in activating fibroblasts, the primary effector cells in fibrosis, mainly by promoting tissue hypoxia and chronic inflammation [17,80,81,82,83]. Vasospasm affecting arterioles and arteries, clinically manifested as Raynaud’s phenomenon, further exacerbates fibroblast activation through ischemia–reperfusion injury, a process involving tissue damage caused by alternating periods of insufficient blood supply and reperfusion [84]. Fibroblasts themselves undergo phenotypic modulation, acquiring a profibrotic phenotype, and exhibit dysregulated responses to inflammatory signals. Notably, they demonstrate excessive ECM synthesis, contributing to the tissue fibrosis [78]. The cellular origins of myofibroblasts in SSc are diverse, encompassing not only resident tissue fibroblasts but also vascular wall-resident endothelial cells and pericytes, epithelial cells, adipocytes, and bone marrow-derived fibrocytes [78]. Myofibroblasts originating from this heterogeneous array of cellular sources collectively orchestrate the pathogenesis of the extensive fibrosis observed in SSc [78]. The preceding discussion elucidates the fundamental pathophysiology of SSc, highlighting a shared mechanistic basis that operates across various organs affected by fibrosis, and thus can be conceptualized as a foundational trans-organ pathophysiology [72,73].

2.6. Fibrosis in SSc

The key effector cells driving SSc fibrosis are α-smooth muscle actin (α-SMA)-positive myofibroblasts, which secrete excessive ECM in affected tissues. These cells derive from a heterogeneous population of precursors, including local fibroblasts, circulating fibrocytes [85], and cells undergoing epithelial–mesenchymal [86], and endothelial–mesenchymal transition (EndoMT) [87,88], and adipocyte–myofibroblast transdifferentiation [89]. This state of fibroblast activation is the final outcome of the SSc pathogenic process, and the cells maintain their activated phenotype through a combination of intrinsic and extrinsic mechanisms.

A primary growth factor driving dermal fibroblast activation in SSc is TGF-β, which induces the production of ECM components like fibrillar collagens (types I, III, and V). Its expression is high in early, active disease and diminishes in established fibrosis. The localization of its isoforms suggests a role in the disease’s inflammatory phase; specifically, TGF-β1 and TGF-β2 are prominently expressed around dermal vessels in connection with perivascular mononuclear infiltrates, while all three isoforms are detectable throughout the ECM [90,91,92]. Consequently, in the early stages of SSc, TGF-β appears to promote inflammation by recruiting leukocytes through the modulation of cell adhesion molecules and the establishment of chemokine gradients, by activating leukocytes, and by inducing various proinflammatory cytokines and mediators. Conversely, in the sclerotic phase, SSc dermal fibroblasts exhibit constitutive activation with a profibrotic phenotype, resembling that of normal fibroblasts treated with TGF-β1, even when TGF-β expression is diminished or undetectable in the skin [93]. This suggests that SSc fibroblasts possess a self-activation system, one at least partially mediated via autocrine TGF-β signaling. The increased expression of latent TGF-β receptors, including integrin αVβ3, integrin αVβ5, and thrombospondin-1, contributes to this process in SSc fibroblasts [94,95,96,97,98]. These receptors recruit and activate latent TGF-β on the cell surface, efficiently increasing the concentration of active TGF-β in the cellular microenvironment. Further expanding on the mechanisms of TGF-β-driven fibrosis, Meng et al. [99] identified ADAM19 as a significantly upregulated metalloproteinase in SSc skin fibrosis, demonstrating its role in promoting TGF-β-induced ECM deposition and fibroblast activation through the shedding of pro-fibrotic neuregulin-1 (NRG1), thereby contributing to the development of skin fibrosis in SSc. Thus, dermal fibroblasts are constitutively activated, at least partially, through autocrine TGF-β signaling, in SSc lesional skin.

SSc dermal fibroblasts respond differently to T-cell stimuli, relative to healthy fibroblasts. Normally, collagen production is downregulated by Th1 cells via IFN-γ [100], and by Th2 cells via membrane-associated tumor necrosis factor-α (TNF-α) [101], which counter the profibrotic effect of IL-4. In SSc, however, fibroblasts are resistant to this suppression, especially that from the Th2 cells [100,101]. One proposed reason is the high secretion of progranulin, a TNF receptor antagonist that negates the anti-fibrotic effect of TNF-α [102]. This specific lack of response to Th2-mediated suppression is thought to be a critical driver of fibroblast activation during the early, Th2-polarized phase of dcSSc. Adding to the complexity of fibroblast activation, Bergmann et al. [103] discovered a mutual amplification loop between GLI2/Hedgehog and JUN/AP-1 signaling pathways within SSc fibroblasts, where these pathways synergistically enhance each other’s activity, resulting in sustained fibroblast activation and collagen production, highlighting a potential target for combined therapeutic interventions.

The interaction is bidirectional, as SSc dermal fibroblasts also shape the differentiation of inflammatory cells. For example, they direct the transdifferentiation of Tregs into Th2-like cells via IL-33 in the skin [30,104]. By overproducing galectin-9, they also inhibit IFN-γ expression in skin-infiltrating CD4+ T cells, which fosters fibrosis within the Th2/Th17-dominant microenvironment [105]. These findings suggest SSc fibroblasts affect skin immunity more extensively than previously thought.

Collectively, SSc fibroblasts perpetuate their activation through autocrine signaling and feedforward loops with other cells, culminating in the irreversible fibrotic remodeling of multiple organs.

3. Verification of Pathogenesis Hypotheses: Insights from Recent Analytical Innovations

Pathogenesis hypotheses of SSc, as previously outlined, have been largely formulated based on conventional clinical and basic research methodologies, utilizing clinical samples from SSc patients (primarily lesional skin and lung biopsy tissues, and peripheral blood) and animal models. However, recent years have witnessed a transformative shift with the advent of cutting-edge analytical techniques, including single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, imaging mass cytometry, and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq), particularly in the context of lesional skin analysis. These advanced technologies have provided unprecedented opportunities for the successive validation and refinement of the aforementioned pathogenesis hypotheses. Representative studies employing these innovative approaches are introduced below.

3.1. Scleroderma-Associated Fibroblast (ScAF) Identification via scRNA-Seq Analysis

In a landmark study in 2022, Gur et al. [106] leveraged scRNA-seq to analyze skin tissues from a large cohort comprising 97 patients with SSc and 56 healthy individuals, subsequently publishing transformative findings. Their comprehensive analysis led to the refined classification of skin fibroblasts into 10 distinct cellular subpopulations. Notably, myofibroblasts, which have historically been considered central effector cells in the pathogenesis of SSc, were found to represent only a minor fraction, constituting approximately 1% of the total cellular population. Conversely, the most abundant fibroblast subpopulation was identified as LGR5 (Leucine-rich repeat-containing G-protein coupled Receptor 5)-positive fibroblasts, which account for approximately 30% of the cells in healthy individuals. Gene expression pattern analysis indicated that this LGR5+ fibroblast population plays a critical homeostatic role in maintaining normal skin architecture. However, a significant reduction in the abundance of this cell population was observed in SSc patients. Furthermore, these Scleroderma-Associated Fibroblasts (ScAFs) exhibited a constellation of molecular characteristics consistent with previously described SSc skin fibroblasts, including the following: 1, pathologically excessive ECM production coupled with suppressed degradation; 2, aberrant activation of Type I interferon signaling, TGF-β pathway, and IL-1 pathway; 3, dysregulation of Wnt signaling and IGF1 signaling, and abnormal expression of CCN1 family proteins; 4, pathologically activated angiogenesis, increased vascular fragility, and enhanced coagulation and platelet aggregation; 5, diminished antioxidation and adipogenesis capacity; and 6, overexpression of CDKN2A (p16) and CDKN1A (p21), indicative of enhanced cellular senescence. Notably, the study also demonstrated a significant inverse correlation between the cellular density of this ScAF population and skin score. These compelling findings suggest that ScAFs, identified as the principal fibroblast subpopulation orchestrating the fibrotic pathology of SSc, may represent promising novel therapeutic targets. Conversely, the investigation also revealed a concomitant increase in vascular endothelial cells and pericytes alongside the progression of skin sclerosis. In particular, RGS5 (Regulator of G-protein Signaling 5)-positive vascular pericytes demonstrated a positive correlation with skin score. These observations may be interpreted as providing support for conventional pathogenesis hypotheses that posit vascular endothelial cells and pericytes as primary cellular origins of pathogenic fibroblasts in SSc.

3.2. Spatial Transcriptomics Analysis

Ma et al. [107] performed an in-depth analysis of SSc pathogenesis using single-cell and spatial transcriptomics. They analyzed scRNA-seq data from skin biopsies of twenty-two SSc patients and eighteen healthy controls, alongside spatial RNA-seq data from four SSc patients, to map disease-associated cells and their interactions within SSc lesions. The study reported four key observations. First, fibroblasts, vascular endothelial cells, and pericytes were diffusely present in fibrotic areas of SSc skin. Second, fibroblasts were classified into seven distinct populations; SFRP2⁺ fibroblasts activated and differentiated into COL8A1⁺ fibroblasts (with myofibroblast features) during fibrosis progression. Third, vascular endothelial cells demonstrated heterogeneity, with categorization into seven distinct subpopulations, including arteriolar endothelial cells (EC2) and activated endothelial cells (EC5). EndoMT maturity served as a differentiating factor among these subpopulations, with EC2 identified as the dominant subpopulation within SSc lesions. Fourth, ligand–receptor network analysis indicated that fibroblast–vascular endothelial cell interactions were most pronounced, with EC2 and COL8A1⁺ myofibroblast-like fibroblasts being the key communicators. These results reinforce existing pathogenesis models implicating vascular wall cells as myofibroblast origins in SSc.

3.3. Integrated Analysis: scRNA-Seq and snATAC-Seq, Focusing on Vascular Endothelial Cells

In their 2024 study, Huang et al. [108] performed a comprehensive analysis using scRNA-seq and snATAC-seq data to investigate SSc vasculopathy. Analyzing skin biopsies from twenty-seven SSc patients and ten healthy controls via scRNA-seq, and snATAC-seq data from eight SSc patients and six controls, they explored the role of transcription factors in SSc-associated vascular pathology. The scRNA-seq analysis of lesional SSc skin revealed two key observations: first, increased apoptosis and decreased cell numbers in arteriolar endothelial cells; and second, an elevation in tip and stalk cell populations, indicative of constitutively enhanced angiogenesis in dermal microvascular endothelial cells. These findings align with established pathogenesis hypotheses of SSc, particularly regarding destructive vasculopathy and angiogenic abnormalities. Furthermore, snATAC-seq analysis indicated increased chromatin accessibility at the ETS motif in SSc vascular endothelial cells, supporting the involvement of ETS transcription factors, especially FLI1, in SSc vasculopathy, consistent with prior research. These integrated analyses using scRNA-seq and snATAC-seq reinforce the conventional understanding of SSc pathogenesis by highlighting the roles of vascular endothelial cell apoptosis, dysregulated angiogenesis, and ETS transcription factors in the development of SSc vasculopathy (Figure 1).

3.4. Vascular Niche Analysis by Spatial Proteomics Using Imaging Mass Cytometry

Rius Rigau et al. [109] employed imaging mass cytometry to conduct a vascular niche analysis in skin samples from 19 SSc patients and 14 healthy individuals. Their spatial proteomics-based approach identified seven subpopulations of vascular endothelial cells based on their unique protein expression profiles. In SSc patients, the researchers reported an increased population of CD34⁺;αSMA⁺;CD31⁺ cells alongside a reduction in vascular endothelial progenitor cells. The perivascular microenvironment of CD34⁺;αSMA⁺;CD31⁺ cells was characterized by a significant presence of immune cells, predominantly CD4⁺ T cells and myeloid cells, as well as myofibroblasts. Moreover, CD34⁺;αSMA⁺;CD31⁺ cells exhibited markers of EndoMT, such as SNAI1, SNAI2, TWIST, and ZEB1. The density of CD34⁺;αSMA⁺;CD31⁺ cells was found to correlate with the clinical progression of skin sclerosis. These observations reinforce the established pathogenesis model wherein vascular endothelial cells contribute to the myofibroblast population through EndoMT in SSc-related fibrosis.

3.5. Novel Pathogenesis Mechanism of SSc Skin Fibrosis Suggested by Epigenetic Analysis Using ATAC-Seq

Liu et al. [110] conducted an epigenetic analysis to explore novel pathogenesis mechanisms in SSc skin fibrosis, employing ATAC-seq. Using flow cytometry, they isolated eight skin-resident cell types—fibroblasts, vascular endothelial cells, epidermal cells, CD4⁺ T cells, CD8⁺ T cells, dendritic cells, Langerhans cells, and macrophages—from the healthy, lesional, and non-lesional SSc skin of seven SSc patients and six healthy controls, totaling 19 samples. Leveraging the known enrichment of disease-susceptibility SNPs in non-coding regulatory DNA, they hypothesized that cell-type-specific chromatin accessibility analysis at SSc-associated SNP loci could pinpoint pathogenic cell types. Their analysis revealed significantly increased chromatin accessibility in SSc-associated SNP regions specifically within dendritic cells (DCs), compared to other skin cell types. Re-analysis of time-series RNA-seq data from SSc lesions further supported this, showing a strong positive correlation between the DC gene signature and skin fibrosis score. Specifically, conventional DCs (cDCs) were identified as a key cellular population. Immunohistochemical validation using ZBTB46, a cDC marker, confirmed significant infiltration of ZBTB46⁺ cells into SSc lesions. These epigenetic findings suggest a previously unappreciated role for cDCs in SSc skin fibrosis, offering a novel perspective on SSc pathogenesis beyond conventional hypotheses. The importance of dendritic cells in the pathogenesis of SSc is also demonstrated in the following articles [111,112]

3.6. Novel Therapeutic Targets Identified by Gene Expression Meta-Analysis of Lung Tissue

Yang et al. [113] published a gene expression meta-analysis of lung tissue, examining 38 patients with SSc-ILD and 18 healthy controls. Their analysis, utilizing three public datasets (GSE48149, GSE81292, GSE76808), identified the activation of epithelial–mesenchymal transition, cellular senescence, coagulation, and DNA repair pathways as characteristic changes in SSc-ILD lung tissue. Consistent with an aging phenotype, telomere length in type II alveolar epithelial cells from SSc-ILD lungs was found to be reduced, indicating enhanced cellular senescence. The current therapeutic development for SSc-ILD primarily targets myofibroblasts and inflammation/autoimmunity. However, this study suggests that cellular senescence and coagulation pathways could offer novel therapeutic avenues for SSc-ILD.

Plasminogen Activator Inhibitor-1 (PAI-1) is a serpin that inhibits tissue-type Plasminogen Activator (tPA) and urokinase-type Plasminogen Activator (uPA), and regulates the plasmin activation and the fibrinolytic system [114]. Emerging research underscores the significance of PAI-1 in regulating cellular senescence, with findings demonstrating that PAI-1 not only serves as a senescence marker but also actively mediates senescence pathways, impacting lifespan and age-related pathologies [115,116,117]. The tPA, uPA, and PAI-1 are thought to play an important role in the maintenance of endothelial homeostasis, and are associated with the endothelial dysfunction of SSc [118,119]. Therefore, PAI-1 inhibition may be therapeutic for SSc-ILD.

4. Organ-Specific Pathophysiology Modifiers: Refining the Landscape of SSc Organ Involvement

To fully elucidate the organ-specific manifestations of SSc, it is essential to consider organ-specific pathophysiology modifiers, in addition to the broadly acting trans-organ basic pathophysiology. The following sections detail the principal organ-specific modifiers across major organ systems affected by SSc [13].

4.1. Cutaneous Pathology

Epidermal cells and adipocytes function as key pathophysiology modifiers in the skin in SSc (Figure 2).

4.1.1. Keratinocytes

The epidermis in SSc is an active participant in the disease process, as evidenced by research identifying the upregulation of numerous molecules in affected skin. These include growth factors (e.g., TGF-β, VEGF, and CTGF), cytokines (e.g., IL-1α, IL-6, and TNF-α), and chemokines (e.g., CCL2, CCL5), in addition to other proteins like endothelin-1, IL-21 receptor, specific keratins, psoriasin, and galectin-7 [120,121,122,123,124,125,126,127,128,129]. As several of these molecules—notably IL-1α, CTGF, and IL-6—have strong pro-fibrotic properties, it is probable that SSc keratinocytes play a role in activating dermal fibroblasts.

Experiments using a recently developed mouse model of SSc have suggested a role for various epithelial cells in the disease’s development, including keratinocytes, esophageal stratified squamous epithelia, and medullary thymic epithelial cells [130]. Deficiency of the transcription factor Fli1, a potential SSc susceptibility factor [14], induces SSc-like properties in various cell types, including fibroblasts, endothelial cells, keratinocytes, T cells, B cells, cDC, and macrophages [17,130,131,132,133,134]. Notably, epithelial cell-specific Fli1 knockout mice (Fli1^flox/flox; K14-Cre^+/− mice), which exhibit SSc-like phenotypic features in epithelial cells, spontaneously develop dermal and esophageal fibrosis due to epithelial cell activation in the skin and esophagus. Furthermore, these mice develop ILD, mediated at least in part by T cells autoreactive to lung antigens, resulting from impaired negative selection and Treg development in the thymus. One component of this impaired central tolerance is attributed to the downregulation of autoimmune regulator (Aire), which modulates the processing and presentation of self-antigens in medullary thymic epithelial cells [135,136]. Importantly, epithelial cell-specific Fli1 knockout mice lacking an acquired immune system (Rag1^−/−; Fli1^flox/flox; K14-Cre^+/− mice) spontaneously develop dermal and esophageal fibrosis, along with mast cell infiltration in the skin, but do not develop ILD [137]. This suggests that epithelial cell activation alone can induce tissue fibrosis through the activation of innate immunity. This novel murine model indicates that abnormally activated epithelial cells underlie selective organ fibrosis and autoimmunity in SSc.

Another potential aspect of the keratinocyte-dependent regulation of dermal fibrosis is the interplay between the immune system and the skin microbiota. This area of research has recently garnered significant attention regarding inflammatory skin diseases, such as atopic dermatitis [138] and SLE [139]. This dialogue begins when keratinocytes detect pathogen-associated molecular patterns by the use of microbes using pattern-recognition receptors. This recognition prompts the keratinocytes to release antimicrobial peptides (AMPs), which can kill or inactivate microorganisms and activate other cells like dermal fibroblasts and endothelial cells [140]. The expression of these AMPs can be either constant or temporary, with the latter being regulated by the skin’s microbial community. The specific impact of the skin microbiota on keratinocyte function and immunity in SSc is not yet established, but the finding of microbiome dysbiosis in affected skin suggests it may be a contributing factor [141].

4.1.2. Adipocytes

Recently, subcutaneous adipose tissue has been increasingly implicated in the development of skin fibrosis in SSc. This is histologically significant, as the skin is characterized by a large, adjacent layer of fat. The concept that adipose tissue could be a source of fibrotic cells is consistent with the knowledge that myofibroblasts can be derived from non-fibroblast precursors in a pro-fibrotic environment [142]. According to lineage-tracing studies [143], subcutaneous adipocytes are highly plastic cells capable of transdifferentiating into myofibroblasts [144]. Indeed, a significant proportion of activated myofibroblasts in SSc-involved skin appear to derive from adipocytes located adjacent to the deep dermis [89,145].

Adipocytes also influence the disease by producing a range of signaling molecules called adipokines [146]. When adipocyte loss or dysfunction alters the balance of these adipokines, it may contribute to the characteristic inflammation, vasculopathy, and fibrosis of SSc [147,148,149,150,151,152,153,154,155,156,157]. The role of adiponectin, a well-studied example, illustrates this connection; its serum and tissue levels are inversely related to skin score in patients [147,156,158]. Moreover, mice lacking adiponectin show reduced dermal fibrosis after bleomycin challenge [159], and AdipoRon, a drug that inhibits adiponectin signaling, lessens SSc-like features in mouse models [160]. Taken together, these findings indicate that subcutaneous adipose tissue is a significant driver of skin fibrosis in SSc.

4.2. Gastrointestinal Pathology

Affecting approximately 90% of patients, gastrointestinal (GI) issues are a leading cause of morbidity in SSc, stemming from impaired motility and deficient enzyme secretion throughout the digestive system [161,162,163]. The esophagus is the most commonly affected site, resulting in a high prevalence (70–90%) of upper GI symptoms like GERD and dysphagia. Following the esophagus, the anorectal region, small bowel, stomach, and colon are also frequently involved. This leads to a variety of lower GI symptoms (seen in 20–70% of patients), which can include small intestinal bacterial overgrowth, malabsorption, diarrhea, pseudo-obstruction, and fecal incontinence [164].

Consistent with observations in the skin and other internal organs, the common SSc-specific pathological cascade broadly impacts the GI system, ultimately leading to extensive atrophy and fibrosis of the gastrointestinal smooth muscle [165]. Additionally, SSc exhibits a GI organ-specific pathology relevant to the complex and highly organized enteric nervous system. Vascular structural changes, such as capillary rarefaction and arteriolar stenosis, induce tissue hypoxia throughout the GI tract, resulting in autonomic axonal degeneration [166]. Thus, SSc-associated GI involvement is attributed to the hypomotility and dysmotility stemming from extensive atrophy and fibrosis of the enteric smooth muscle, as well as disturbances in the enteric nervous system. Indeed, SSc-associated esophageal dysfunction comprises three pathological components: (i) reduced lower esophageal sphincter pressure; (ii) ineffective esophageal body peristalsis, particularly in the lower esophagus; and (iii) discoordination of peristaltic and lower esophageal sphincter function [161,165,167,168,169]. Ultimately, the long-term course of SSc-associated GI involvement often culminates in atrophic and fibrotic changes within the gastrointestinal smooth muscle. The precise temporal dynamics and predisposing factors associated with this progression are complex, with recent evidence suggesting that while esophageal dysfunction can occur early, the overall worsening of GI symptoms over extended periods may be influenced more by characteristics like patient sex and specific autoantibody profiles, such as ACA, rather than strictly by cutaneous subtypes [170].

The neuropathy in SSc-related GI dysfunction may be partly driven by pathogenic autoantibodies. For instance, some patients have antibodies against myenteric neurons [171], including those specific for the muscarinic acetylcholine receptor M3 [172,173]. These are considered pathogenic because they disrupt peristalsis in animal experiments [171,172], and in SSc patients, their levels are associated with more severe GI disease [174], suggesting a pathogenic role for these antibodies in humans. Consistent with this, the motility of the pharynx and proximal esophagus, which is regulated by the somatic nervous system, remains normal in SSc [174,175]. Overall, current evidence supports the concept that a combination of autoimmunity, vasculopathy, and fibrosis underlies GI involvement in SSc.

Drawing a parallel with the skin, the esophagus’s stratified squamous epithelia may directly drive fibrosis in SSc. This idea is supported by the Fli1^flox/flox; K14-Cre^+/− mice, where the esophagus displays SSc-like molecular features, including increased IL-8 and prominent IL-1β expression. This epithelial dysfunction could also explain symptoms, as epithelium-derived cytokines are thought to cause refractory GERD-related and functional heartburn [176]. This is particularly relevant, because SSc patients’ symptoms often do not correlate with objective physiological findings [161,177]. Thus, an abnormal epithelial phenotype is a plausible, though not yet proven, contributor to both fibrosis and heartburn in SSc.

The gut microbiota is also a key component of GI-specific pathology in SSc. It is known to modulate the immune system and is implicated in autoimmune diseases through immune dysfunction [178,179,180]. In SSc, studies show that the intestinal microbial composition is different from that in healthy individuals, with a decrease in commensal bacteria (e.g., Faecalibacterium, Clostridium) and an increase in pathobionts (e.g., Fusobacterium) [181,182]. It is not yet clear whether these changes are a cause or a consequence of SSc or its treatments. However, a report that fecal microbiota transplantation reduced lower GI symptoms suggests a direct role [183]. More research is needed to clarify the mechanisms by which the gut microbiota interacts with inflammatory and fibrotic pathways in SSc.

In conclusion, the abnormally activated stratified squamous epithelia and enteric nervous system dysfunction constitute organ-specific pathological processes within the GI tract in SSc (Figure 3).

4.3. Pulmonary Pathology

Pulmonary involvement, encompassing ILD and pulmonary hypertension (PH), represents the primary cause of SSc-related mortality [184,185]. ILD in SSc arises from the common SSc-specific pathological cascade and can be further influenced by microaspiration of gastric contents due to GERD. Pulmonary arterial hypertension (PAH) in SSc is attributed to pulmonary arteriolar stenosis resulting from occlusive vascular fibrosis.

The World Health Organization (WHO) classification categorizes PH into five groups: Group 1, PAH characterized by pre-capillary pulmonary vasculopathy involving small pulmonary arterioles; Group 2, PH due to left heart disease; Group 3, PH due to lung diseases and/or hypoxia (including ILD); Group 4, PH due to pulmonary artery obstructions (e.g., chronic thromboembolic pulmonary hypertension, CTEPH); and Group 5, PH with unclear multifactorial mechanisms [186]. SSc-associated pulmonary hypertension (SSc-PH) most commonly falls into Group 1 (PAH), Group 2, or Group 3 [187,188]. Additionally, SSc patients with associated antiphospholipid antibody syndrome may be at risk for developing Group 4 PH. It has been observed that PAH (Group 1) is the most frequent form of PH when associated with connective tissue diseases like SSc [189]. These distinct pathologies, particularly PAH and ILD, can indeed coexist to varying extents in SSc patients. The presence of concomitant ILD in PAH-SSc patients has been shown to worsen hemodynamics and pulmonary function tests, making the clinical classification and management more challenging [189]. Such coexistence of PAH and ILD is a significant concern, as these are the two leading causes of mortality in SSc [189].

Historically, SSc-PAH was considered more prevalent in patients with limited cutaneous SSc (lcSSc) and those with ACA [190,191]. However, the understanding of these associations continues to evolve with larger and more contemporary cohort studies. For instance, a meta-analysis by Rubio-Rivas et al. [192] reported an overall PAH prevalence in SSc of 6.4%, with a prevalence of 7.7% in lcSSc and 6.3% in dcSSc, supporting a higher prevalence in lcSSc. Survival for patients with Group 1 SSc-PAH has shown improvement in the most recent decade, potentially due to earlier detection through screening programs and more effective therapeutic strategies, including upfront combination therapy [188,193].

In a more focused sense, SSc-PAH is histologically characterized by a proliferative and obliterative vasculopathy predominantly affecting the pulmonary arterioles. This is considered a consequence of the shared SSc-specific pathological cascade involving endothelial dysfunction, inflammation, and fibrotic remodeling [190]. The vascular lesions in SSc-PAH often feature perivascular lymphocytic infiltrates and significant intimal fibrosis. Notably, classical plexiform lesions, which are characteristic of idiopathic PAH, are less commonly observed in SSc-PAH [190].

Furthermore, the clinical picture of SSc-PAH can be frequently complicated by, or mimicked by, conditions such as pulmonary veno-occlusive disease (PVOD) or PAH with overt features of venous/capillaries involvement. PVOD is also classified under WHO Group 1 PH but represents a distinct pathological entity primarily affecting pulmonary venules and capillaries. PVOD arising in a background of connective tissue disease such as SSc (PVOD-like changes) is a rare but critical condition, often underdiagnosed in SSc due to its symptomatic similarity to PAH. A key distinguishing concern with regard to PVOD-like changes is its particularly poor prognosis and the significant risk of precipitating acute pulmonary edema with the use of PAH-specific vasodilator therapies. Therefore, careful diagnostic evaluation is paramount. High-resolution computed tomography (HRCT) plays a vital role when PVOD-like changes is suspected, with characteristic findings including centrilobular ground-glass opacities, interlobular septal thickening, and mediastinal lymph node enlargement [187,194].

Therefore, understanding and managing SSc-PH necessitates a comprehensive approach that considers all components of the pulmonary circulation. Given that SSc is a disease characterized by multi-organ involvement, a thorough evaluation of all elements related to pulmonary circulation is essential when addressing PH in these patients (Figure 4).

ILD is detectable in 50–60% of SSc patients via HRCT [195,196]. Risk factors for the development of SSc-ILD encompass dcSSc [197], African American ethnicity [198], shorter disease duration [199], older age at disease onset [197], and the presence of anti-topoisomerase I antibody and/or absence of ACA [197]. ILD typically manifests early in the course of dcSSc, particularly within the first 3 years of disease onset [197,199,200], while in lcSSc patients, ILD can arise at any point during the disease course [201]. The clinical trajectory of SSc-ILD is heterogeneous; some patients maintain stable forced vital capacity (FVC), whereas others experience a progressive decline in pulmonary function [202]. ILD progression is generally most pronounced within the initial 4 years following SSc onset, subsequently slowing or ceasing entirely, even without therapeutic intervention [203]. Severe ILD, defined by an FVC decline below 50%, is reported to affect approximately 15% of the total SSc population [203,204]. The predominant histological pattern in SSc-ILD is nonspecific interstitial pneumonia (NSIP), observed in roughly two-thirds of patients [205]. Usual interstitial pneumonia (UIP) is present in a smaller proportion of SSc-ILD cases [205,206,207] and may correlate with less favorable prognoses [208].

Histologically, nonspecific interstitial pneumonia (NSIP) in SSc-ILD is categorized into four stages [209]:

• * Stage 1 (Initial): Characterized by microvessel overdevelopment with structural abnormalities, and alveolar septal thickening with numerous α-SMA-positive myofibroblasts. The overdeveloped microvessels contain blood cells within their lumina, indicating maintained functional circulation.
• * Stage 2 (Progressive ECM Deposition): Marked by substantial and progressive ECM deposition, irregular and indistinct alveolar septal borders, further structural disorganization of microvessels, and obliteration of larger blood vessels. Disarray or partial loss of the alveolar epithelium is also evident.
• * Stage 3 (Extensive Fibrosis): Progression of fibrosis extensively damages vital lung structures, including alveoli and vasculature.
• * Stage 4 (Final): The lung transforms into a contracted fibrous organ devoid of alveoli and vasculature.

The early microvascular alterations and subsequent progressive fibrotic changes reinforce the concept that SSc-ILD is driven by the common SSc-specific pathological cascade, similar to other organ involvements.

Above pathological findings suggest the presence of foundational trans-organ pathophysiology in the lung tissue.

Microaspiration of gastric contents due to GERD is a potential factor driving the progression of SSc-ILD. Clinical data, histological analyses, and animal studies support this hypothesis. Several clinical studies have shown a positive correlation between increased lung fibrosis severity and more frequent reflux episodes, as well as greater proximal extension of refluxate [210]. In a rat GERD model, pulmonary parenchymal fibrosis was induced by introducing gastric content into the lungs [211]. Analysis of lung biopsy specimens identified a distinct histological pattern of lung disease, centrilobular fibrosis (CLF), particularly prevalent in SSc patients with severe GERD [212]. CLF is characterized by a predominantly bronchocentric distribution of lesions and the presence of intraluminal basophilic material and foreign bodies within the bronchi, sometimes accompanied by multinucleated giant cell reactions. In a prior study examining open lung biopsies from 22 SSc-ILD patients [213], isolated CLF was observed in 21% of cases, and a CLF pattern was present in 84% of patients with a predominant NSIP pattern, suggesting that GERD may exacerbate underlying NSIP in SSc-ILD. Although clinical trial data have not yet demonstrated pulmonary function improvement in SSc-ILD following aggressive GERD management, aggressive GERD treatment may still benefit the majority of SSc patients. Overall, GERD acts as an organ-specific disease modifier in SSc-ILD (Figure 4).

4.4. Cardiovascular Pathology

Histological examination of autopsy tissues from SSc patients without prior clinical cardiac symptoms reveals evidence of myocardial disease in all cases [214]. Thus, cardiac involvement is nearly ubiquitous in SSc patients, although often clinically silent [215,216]. Once clinically manifest, however, cardiac involvement carries a poor prognosis [215,217,218,219]. Primary cardiac involvement in SSc encompasses a wide spectrum of clinical manifestations, including arrhythmias, conduction system defects, myocarditis, pericarditis, systolic and diastolic ventricular dysfunction, and heart failure [220,221]. Primary myocardial involvement is estimated to account for approximately 30% of deaths in SSc patients [185,219,222].

In a study employing cardiovascular magnetic resonance (CMR) parametric mapping in SSc patients, Purevsuren et al. [223] demonstrated that native T1 mapping effectively detects early myocardial changes and correlates with left-ventricular diastolic dysfunction, with more pronounced myocardial involvement observed in dcSSc compared to lcSSc. This finding of early diffuse myocardial edema-like lesions on contrast-enhanced MRI mirrors the edematous induration seen in early skin lesions of SSc, suggesting a parallel pathological process, affecting both skin and internal organs, in the initial stages of the disease.

Although the precise molecular mechanisms of SSc-related cardiomyopathy remain incompletely understood [220,224,225,226], the prevailing consensus attributes a central role to microvascular disease. The proposed mechanism is that structural microvascular defects, including capillary rarefaction and arteriolar stenosis, result in tissue hypoxia. This oxygen deprivation is believed to subsequently trigger inflammation and excessive ECM synthesis by cardiac fibroblasts [215]. This model is supported by histological findings from SSc autopsy specimens, which show increased inflammation, vascular damage, and ECM deposition compared to controls [214]. The microvascular origin is further indicated by the patchy, non-coronary distribution of fibrosis throughout the ventricles [224,227].

In addition to structural damage, abnormal vasoreactivity of small cardiac vessels, known as “myocardial Raynaud’s phenomenon,” contributes to cardiac involvement in SSc. This concept is supported by multiple lines of evidence. For instance, cold exposure can induce this phenomenon in approximately 30% of SSc patients with a history of Raynaud’s, and the effect is preventable with calcium channel blockers (CCBs) [228]. The therapeutic benefit of vasodilators like nifedipine, nicardipine, and captopril, which acutely improve myocardial perfusion and function [229,230,231,232], also points to a vasospastic component. Finally, the absence of prior CCB therapy is an independent factor associated with left-ventricular dysfunction.

Thus, from a management perspective, the cardiac involvement in SSc must be viewed as being a result of two distinct categories of vascular change. One category is structural, encompassing capillary rarefaction and arteriolar stenosis, while the other is functional, specifically the myocardial Raynaud’s phenomenon.

4.5. Scleroderma Renal Crisis

One frequent form of renal involvement in SSc patients is a subclinical renal vasculopathy, characterized by vascular damage and normal renal function [233]. Scleroderma renal crisis (SRC) complicates the course of 5–10% of SSc patients [234,235,236,237], presenting most commonly within the first few years of disease onset, particularly in those with dcSSc [234,236]. Key risk factors include rapidly progressive skin thickening, the presence of anti-RNA polymerase III antibodies [234,236,238,239,240], and recent high-dose corticosteroid therapy (>15 mg/day) [234,235,236,237,241,242]. While strongly associated with anti-RNA polymerase III antibodies, SRC can rarely occur in patients with lcSSc, including those positive for ACA [239,241,243,244].

Clinically, SRC typically manifests suddenly, with an abrupt onset of accelerated hypertension (often > 150/85 mmHg or a significant rise from baseline) and acute kidney injury (AKI), frequently accompanied by headache, visual changes, or signs of hypertensive encephalopathy or cardiopulmonary failure [234,236,245]. Approximately 10–11% of cases manifest as normotensive SRC, which may be associated with corticosteroid use and has been linked to a poorer prognosis, possibly due to delayed diagnosis or a potentially more severe underlying pathology [234,236,241,245]. Laboratory findings may include microangiopathic hemolytic anemia (MAHA) and thrombocytopenia, features overlapping with hemolytic uremic syndrome (HUS)/thrombotic microangiopathy (TMA) [234,236,241,245].

The core pathogenesis involves endothelial injury in renal arterioles, leading to intimal accumulation of myxoid material, subsequent intimal proliferation resulting in luminal narrowing (”onion-skin” lesions), thrombosis, and fibrinoid necrosis [234,236,241,245]. Renal biopsy findings, such as the extent of vascular thrombosis, severe glomerular ischemic collapse, and peritubular capillary C4d deposits, may predict failure to recover renal function [245]. This vascular damage triggers activation of the renin–angiotensin–aldosterone system (RAAS), causing hyperreninemia and creating a vicious cycle of worsening hypertension and renal ischemia [234,236,241]. Renal vasospasm (“renal Raynaud’s”) is also thought to contribute significantly to the reduced renal perfusion [234,236]. Historically, it has been reported that SSc patients already on angiotensin-converting enzyme (ACE) inhibitor therapy at the time of SRC onset may experience worse outcomes [236,246,247]. More recently, studies have indicated that prior exposure to ACE inhibitors might not only be associated with poorer SRC prognosis, but could also represent an independent risk factor for the development of SRC itself, particularly in hypertensive SSc patients [248]. These findings have led to the recommendation against the prophylactic use of ACE inhibitors to prevent SRC in patients, without a clear indication for these drugs, such as established hypertension [236,247]. This evolving understanding of the complex relationship between ACE inhibitor use and SRC may offer new insights into the underlying pathogenetic mechanisms of this severe SSc complication.

SRC, along with DUs and SSc-PAH, is considered a manifestation of the systemic, non-organ-specific vasculopathy underlying SSc, primarily driven by arteriolar stenosis affecting different vascular territories [13].

5. Conclusions

The current understanding of SSc pathogenesis, bolstered by advanced analytical methods, has been the focus of this review. The skin’s frequent affliction and ease of sampling have made it a cornerstone for pathogenetic investigations. Future research, extending these sophisticated analytical tools to internal organs like the lungs, holds considerable promise for refining our comprehension of the fundamental, widespread pathophysiology and the specific factors that modulate organ involvement in SSc. This improved insight is vital for tailoring more effective treatments, and thereby enhancing disease management and patient prognosis and opening new therapeutic avenues. It is acknowledged that SSc treatment strategies are varied, adapt to specific pathological manifestations, and are the subject of numerous clinical investigations; however, these are not detailed here, and specialized reviews can offer further information [249,250,251,252].

Central to deciphering SSc’s complex cross-organ impact is the identification of its core pathogenic driver. Although immune dysfunction is characteristic of its autoimmune nature and “sclerosis” underscores fibrosis, we propose vasculopathy as the pivotal intermediary linking aberrant immunity to fibrotic outcomes. This view is informed by clinical patterns in which vascular alterations are often the first indicators of SSc, as seen with Raynaud’s phenomenon, and potentially represent the final opportunity for impactful therapeutic modulation, contrasting with early immune dysregulation, which may be subclinical, and established fibrosis, which is typically irreversible.