Search Results (81)

Background/Objectives: Neovascularization, defined as the sprouting of new blood vessels from pre-existing vasculature, is a critical pathological feature in ocular diseases such as pathological myopia and represents a leading cause of corneal vision loss. Vascular endothelial growth factor A (VEGFA) plays a pivotal role in endothelial cell proliferation, migration, survival by anti-apoptotic signaling, and vascular permeability. Dysregulation of VEGFA is closely linked to pathological neovascularization. Exosomes, nanosized phospholipid bilayer vesicles ranging from 30 to 150 nm, have emerged as promising gene delivery vehicles due to their intrinsic low immunogenicity, superior cellular uptake, and enhanced in vivo stability. This study aimed to investigate whether highly purified mesenchymal stem cell (MSC)-derived exosomes loaded with VEGFA siRNA labeled with FAM can effectively suppress pathological corneal neovascularization (CNV) via targeeted cellular transduction and VEGFA inhibition. Furthermore, we examined whether the therapeutic effect involves the modulation of the PI3K–Akt–Caspase-3 signaling axis. Methods: Exosomes purified by chromatography were characterized by electronmicroscopy, standard marker immunoblotting, and nanoparticle tracking analysis. In vitro, we assessed exosome uptake and cytoplasmic release, suppression of VEGFA mRNA/protein, cell viability, and apoptosis. In a mouse CNV model, we evaluated tissue reach and stromal retention after repeated intrastromal injections; anterior segment angiogenic indices; CD31/VEGFA immunofluorescence/immunoblotting; phosphorylated PI3K and Akt; cleaved caspase-3; histology (H&E); and systemic safety (liver, kidney, and spleen). Results: Exosomes were of high quality and showed peak efficacy at 48 h, with decreased VEGFA mRNA/protein, reduced viability, and increased apoptosis in vitro. In vivo, efficient delivery and stromal retention were observed, with accelerated inhibition of neovascularization after Day 14 and maximal effect on Days 17–19. Treatment reduced CD31 and VEGFA, decreased p-PI3K and p-Akt, and increased cleaved caspase-3. Histologically, concurrent reductions in neovascularization, inflammatory cell infiltration, and inflammatory epithelial thickening were observed, alongside a favorable systemic safety profile. Conclusions:VEGFA siRNA-loaded exosomes effectively reduce pathological CNV via a causal sequence of intracellular uptake, cytoplasmic release, targeted inhibition, and phenotypic suppression. Supported by consistent PI3K–Akt inhibition and caspase-3–mediated apoptosis induction, these exosomes represent a promising local gene therapy that can complement existing antibody-based treatments. Full article

It is well established that vascular dysfunction is common in people with diabetes mellitus and is associated with increased risk of heart attack, ischaemic stroke and peripheral vascular disease. Although our understanding of the molecular mechanisms responsible is incomplete, persistent hyperglycaemia observed in poorly controlled diabetes has long been thought to be a critical factor. Multiple studies have, therefore, investigated the effects of poor glycaemic control on vascular function in multiple experimental settings, from in vitro and ex vivo models of primary human cells and tissues through to pre-clinical models. This review consolidates our current understanding of how metabolic and cell signalling pathways triggered by poor glycaemic control, impact vascular function in diabetes. We also evaluate how these pathways could be exploited to develop targeted therapeutic approaches to improve cardiovascular outcomes specifically in people with diabetes. Full article

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is essential for physiological processes such as development and wound healing, but its dysregulation contributes to a range of pathological conditions including cancer, diabetic retinopathy, and chronic inflammation. In recent years, marine-derived compounds have emerged as promising multitarget agents with anti-angiogenic potential. Posidonia oceanica, a Mediterranean seagrass traditionally used in folk medicine, is increasingly recognized for its pharmacological properties, including antioxidant, anti-inflammatory, and anti-invasive activities. This study investigated the effects of a hydroethanolic extract from P. oceanica leaves (POE) on human Endothelial Colony-Forming Cells (ECFCs), a subpopulation of endothelial progenitor cells with high proliferative and vessel-forming capacity, and a relevant model for studying pathological angiogenesis. ECFCs were treated with POE (4–8 µg/mL), and cell viability, morphology, migration, invasion, tube formation, oxidative stress, and activation markers were evaluated. POE did not alter ECFC morphology or viability, as confirmed by Trypan Blue and MTT assays. However, functional assays revealed that POE significantly impaired ECFC migration, invasion, and in vitro angiogenesis in a dose-dependent manner. Under VEGF (Vascular endothelial growth factor) stimulation, POE reduced intracellular ROS accumulation and downregulated key redox-regulating genes (hTRX1, hTRX2, PRDX2, AKR1C1, AKR1B10). Western blot analysis showed that POE inhibited VEGF-induced phosphorylation of KDR, mTOR and p-ERK, while p-AKT remained elevated, indicating selective disruption of VEGF downstream signaling. Furthermore, POE reduced the expression of pro-inflammatory and pro-coagulant markers (VCAM-1, ICAM-1, TF) and partially reversed TNF-α–induced endothelial activation. These findings suggest that POE exerts anti-angiogenic effects through a multitargeted mechanism, supporting its potential as a natural therapeutic agent for diseases characterized by aberrant angiogenesis. Full article

Background: Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are critical conditions lacking effective pharmacologic therapies. Lipopolysaccharide (LPS), a bacterial endotoxin, is a well-established trigger of ALI. Emerging evidence suggests that heparin derivatives may attenuate lung injury, but their mechanisms remain unclear. Methods: This study evaluated the protective effects of 2-O, 3-O desulfated heparin (ODSH) in a murine model of LPS-induced ALI. Mice received LPS intratracheally with or without ODSH pre-treatment. Lung injury was assessed by bronchoalveolar lavage fluid (BALF) analysis, Evans blue dye albumin EBDA) extravasation, and histopathology. Results: ODSH treatment significantly reduced BALF protein concentration, inflammatory cell infiltration, and EBDA leakage. ODSH preserved endothelial barrier function in vitro, as evidenced by transendothelial electrical resistance (TER) measurements in human lung microvascular endothelial cell (HLMVEC) monolayers. Histological assessment (H&E staining) and myeloperoxidase (MPO) staining demonstrated reduced lung injury and neutrophil infiltration in the ODSH group. ODSH also downregulated pro-inflammatory mediators (NF-κB, IL-6, p38 MAPK) and upregulated the anti-inflammatory cytokine IL-10. Conclusions: ODSH mitigates LPS-induced ALI by reducing vascular permeability, neutrophilic inflammation, and pro-inflammatory signaling while enhancing IL-10 expression. These findings suggest ODSH may offer a novel therapeutic approach for treating ALI. Full article

Sclerostin, encoded by the SOST gene, is a novel bone anabolic target for bone diseases. Humanized anti-sclerostin antibody, romosozumab, was approved for treatment of postmenopausal osteoporosis by the US Food and Drug Administration (FDA), but with a black-box warning on cardiovascular risk. The clinical data regarding cardiovascular events from various pre-marketing and post-marketing studies of romosozumab were inconsistent. Overall, the cardiovascular risk of sclerostin inhibition could not be excluded. The restriction of romosozumab in patients with cardiovascular disease history would be necessary. Moreover, genome-wide association study (GWAS) analyses of SOST variants revealed inconsistent results of the association between SOST variations and cardiovascular diseases. Further research incorporating larger sample sizes and functional analyses are necessary. In analyses of serum/tissue sclerostin levels in patients with cardiovascular diseases, the results were controversial but indicated an association between sclerostin and the presence/severity/outcomes of cardiovascular diseases. Nonclinical studies in rodents indicated the inhibitory effect of sclerostin on inflammation, aortic aneurysm, atherosclerosis, and vascular calcification. Sclerostin loop3 participated in the inhibitory effect of sclerostin on bone formation, while the cardiovascular protective effect of sclerostin was independent of sclerostin loop3. Macrophagic sclerostin loop2–apolipoprotein E receptor 2 (ApoER2) interaction participated in the inhibitory effect of sclerostin on inflammation in vitro. Sclerostin in human aortic smooth muscle cells participated in the reduction in calcium deposition. The role of sclerostin in cardiovascular system deserves further investigation. Full article

Retinal ischemic disorders present significant threats to vision, characterized by inadequate blood supply oxygen–glucose deprivation (OGD), oxidative stress, and cellular injury, often resulting in irreversible injury. Catalpol, an iridoid glycoside derived from Rehmannia glutinosa, has demonstrated antioxidative and neuroprotective effects. This study aimed at investigating the protective effects and mechanisms of catalpol against oxidative stress or OGD in vitro and retinal ischemia in vivo, focusing on the modulation of key biomarkers of retinal ischemia, including HIF-1α, vascular endothelial growth factor (VEGF), angiopoietin-2, MCP-1, and the Wnt/β-catenin pathway. Cellular viability was assessed using retinal ganglion cell-5 (RGC-5) cells cultured in DMEM; a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. H₂O₂ (1 mM)/OGD was utilized. Vehicle or different catalpol concentrations were administered 15 min before the ischemic-like insults. The Wistar rat eyes’ intraocular pressure was increased to 120 mmHg for 60 min to induce retinal ischemia. Intravitreous injections of catalpol (0.5 or 0.25 mM), Wnt inhibitor DKK1 (1 μg/4 μL), anti-VEGF Lucentis (40 μg/4 μL), or anti-VEGF Eylea (160 μg/4 μL) were administered to the rats’ eyes 15 min before or after retinal ischemia. Electroretinogram (ERG), fluorogold retrograde labeling RGC, Western blotting, ELISA, RT-PCR, and TUNEL were utilized. In vitro, both H₂O₂ and OGD models significantly (p < 0.001/p < 0.001; H₂O₂ and OGD) induced oxidative stress/ischemic-like insults, decreasing RGC-5 cell viability (from 100% to 55.14 ± 2.19%/60.84 ± 4.57%). These injuries were insignificantly (53.85 ± 1.28% at 0.25 mM)/(63.46 ± 3.30% at 0.25 mM) and significantly (p = 0.003/p = 0.012; 64.15 ± 2.41%/77.63 ± 8.59% at 0.5 mM) altered by the pre-administration of catalpol, indicating a possible antioxidative and anti-ischemic effect of 0.5 mM catalpol. In vivo, catalpol had less effect at 0.25 mM for ERG amplitude ratio (median [Q1, Q3] 14.75% [12.64%, 20.48%]) and RGC viability (mean ± SE 63.74 ± 5.13%), whereas (p < 0.05 and p < 0.05) at 0.5 mM ERG’s ratio (35.43% [24.35%, 43.08%]) and RGC’s density (74.34 ± 5.10%) blunted the ischemia-associated significant (p < 0.05 and p < 0.01) reduction in ERG b-wave amplitude (6.89% [4.24%, 10.40%]) and RGC cell viability (45.64 ± 3.02%). Catalpol 0.5 mM also significantly protected against retinal ischemia supported by the increased amplitude ratio of ERG a-wave and oscillatory potential, along with recovering a delayed a-/b-wave response time ratio. When contrasted with DKK1 or Lucentis, catalpol exhibited similar protective effects against retinal ischemia via significantly (p < 0.05) blunting the ischemia-induced overexpression of β-catenin, VEGF, or angiopoietin-2. Moreover, ischemia-associated significant increases in apoptotic cells in the inner retina, inflammatory biomarker MCP-1, and ischemic indicator HIF-1α were significantly nullified by catalpol. Catalpol demonstrated antiapoptotic, anti-inflammatory, anti-ischemic (in vivo retinal ischemia or in vitro OGD), and antioxidative (in vitro) properties, counteracting retinal ischemia via suppressing upstream Wnt/β-catenin and inhibiting downstream HIF-1α, VEGF, and angiopoietin-2, together with its decreasing TUNEL apoptotic cell number and inflammatory MCP-1 concentration. Full article

The engraftment of transplanted islets depends on the rapid establishment of a novel vascular network. The present study evaluated the effects of cord blood-derived blood outgrowth endothelial cells (BOECs) on the viability of neonatal porcine islets (NPIs) and the post-transplant outcome of grafted NPIs. Dispersed NPIs and human BOECs were reaggregated on microwell cell culture plates and tested for their anti-apoptotic and pro-angiogenic capacity by qRT-PCR and immunohistochemistry. The in vivo functionality was analyzed after transplantation into diabetic NOD-SCID IL2rγ^−/− (NSG) mice. The spheroids, which contained reaggregated neonatal porcine islet cells (REPIs) and BOECs, exhibited enhanced viability and a significantly elevated gene expression of VEGFA, angiopoetin-1, heme oxygenase-1, and TNFAIP3 (A20) in vitro. The development of normoglycemia was significantly faster in animals transplanted with spheroids in comparison to the only REPI group (median 51.5 days versus 60 days) (p < 0.05). Furthermore, intragraft vascular density was substantially increased (p < 0.01). The co-transplantation of prevascularized REPI-BOEC spheroids resulted in superior angiogenesis and accelerated in vivo function. These findings may provide a novel tool to enhance the efficacy of porcine islet xenotransplantation. Full article

Intravenously transplanted mesenchymal stromal cells (MSCs) have been shown to interact with endothelial cells and to migrate to tissues. However, intracellular signals regulating MSC migration are still incompletely understood. Here, we analyzed the role of Rap1 GTPase in the migration of human bone marrow-derived MSCs in vitro and in short-term homing in mice in vivo. MSCs expressed both Rap1A and Rap1B mRNAs, which were downregulated after treatment with siRNA against Rap1A and/or B. In a flow chamber model with pre-established human umbilical vein endothelial cells (HUVECs), Rap1A/B downregulated MSCs interacted for longer distances before arrest, indicating adhesion defects. CXCL12-induced adhesion of MSCs on immobilized Vascular Cell Adhesion Molecule (VCAM)-1 was also decreased after the downregulation of Rap1A, Rap1B, or both, as was CXCL12-induced transwell migration. In a competitive murine short-term homing model with i.v. co-injection of Rap1A+B siRNA-treated and control MSCs that were labeled with PKH 26 and PKH 67 fluorescent dyes, the Rap1A+B siRNA-treated MSCs were detected at increased frequencies in blood, liver, and spleen compared to control MSCs. Thus, Rap1 GTPase modulates the adhesion and migration of MSCs in vitro and may increase the bio-availability of i.v.-transplanted MSCs in tissues in a murine model. Full article

Neurofibromatosis is a genetic disorder arising de novo or with an autosomal dominant transmission that typically presents either at birth or in early childhood, manifesting through distinctive clinical features such as multiple café-au-lait spots, benign tumors in the skin, bone enlargement, and deformities. This literature review aims to resume the spectrum of maternal and fetal complications encountered in pregnant women with neurofibromatosis type 1 (NF1). Thorough research was conducted on databases such as Web of Science, PubMed, Science Direct, Google Scholar, and Wiley Online Library. This review includes 48 case reports, original studies, and reviews on NF1 in pregnancy. The research on the interlink between NF1 and fertility and its influence on human-assisted reproduction techniques is limited. Preimplantation testing (by in vitro fertilization) and prenatal diagnosis (by chorionic villus sampling or amniocentesis) are available to detect affected fetuses. However, genotype–phenotype correlation is difficult to predict. Preconceptional planning and targeted investigations are crucial in understanding the extent of maternal disease. Although in some cases lesions can evolve rapidly during pregnancy, most pregnancies and births in NF1 go well with careful planning. There is a higher incidence of pheochromocytomas and pre-eclampsia, vascular rupture, and cardio-respiratory issues. Anesthesia at birth is a challenge in most cases, and before offering spinal anesthesia, imaging tests should be performed to characterize spinal lesions. General anesthesia may also be challenging when the disease affects the face, neck, upper spine, or airways. Birth-related difficulties may arise because of large neurofibromas located at the level of skin incision or birth canal; uterine atony may be expected if there are uterine lesions. Some complications can develop in postpartum, and affected women should be carefully followed even after pregnancy. Fetal risks include preterm birth (spontaneous or iatrogenic), growth restriction and developmental issues, birth complications, cardiovascular risk, and fetal/neonatal demise. Pregnancies in women with NF1 should be regarded as high-risk and followed in a multidisciplinary fashion. Careful assessment of lesions is of utmost importance before and during pregnancy for anticipating potential maternal risks and before birth to plan anesthesia and delivery. Full article

Cardiovascular diseases, particularly ischemic heart disease, area leading cause of morbidity and mortality worldwide. Myocardial infarction (MI) results in extensive cardiomyocyte loss, inflammation, extracellular matrix (ECM) degradation, fibrosis, and ultimately, adverse ventricular remodeling associated with impaired heart function. While heart transplantation is the only definitive treatment for end-stage heart failure, donor organ scarcity necessitates the development of alternative therapies. In such cases, methods to promote endogenous tissue regeneration by stimulating growth factor secretion and vascular formation alone are insufficient. Techniques for the creation and transplantation of viable tissues are therefore highly sought after. Approaches to cardiac regeneration range from stem cell injections to epicardial patches and interposition grafts. While numerous preclinical trials have demonstrated the positive effects of tissue transplantation on vasculogenesis and functional recovery, long-term graft survival in large animal models is rare. Adequate vascularization is essential for the survival of transplanted tissues, yet pre-formed microvasculature often fails to achieve sufficient engraftment. Recent studies report success in enhancing cell survival rates in vitro via tissue perfusion. However, the transition of these techniques to in vivo models remains challenging, especially in large animals. This review aims to highlight the evolution of cardiac patch and stem cell therapies for the treatment of cardiovascular disease, identify discrepancies between in vitro and in vivo studies, and discuss critical factors for establishing effective myocardial tissue regeneration in vivo. Full article

This study focuses on enhancing controllable fibrin-based hydrogels for tissue engineering, addressing existing weaknesses. By integrating a novel copolymer, we improved the foundation for cell-based angiogenesis with adaptable structural features. Tissue engineering often faces challenges like waste disposal and nutrient supply beyond the 200 µm diffusion limit. Angiogenesis breaks through this limitation, allowing the construction of larger constructs. Our innovative scaffold combination significantly boosts angiogenesis, resulting in longer branches and more capillary network junctions. The copolymer attached to fibrin fibers enables precise adjustment of hydrogel mechanical dynamic properties for specific applications. Our material proves effective for angiogenesis, even under suppression factors like suramin. In our study, we prepared fibrin-based hydrogels with and without the copolymer PVP12400-co-GMA10mol%. Using a co-culture system of human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), we analyzed angiogenetic behavior on and within the modified hydrogels. Capillary-like structures were reproducibly formed on different surfaces, demonstrating the general feasibility of three-dimensional endothelial cell networks in fibrin-based hydrogels. This highlights the biomaterial’s suitability for in vitro pre-vascularization of biohybrid implants. Full article

For reliable in silico or in vitro investigations in, for example, biosensing and drug delivery applications, accurate models of tumor vascular networks down to the capillary size are essential. Compared to images acquired with conventional medical imaging techniques, digitalized histological tumor slices have a higher resolution, enabling the delineation of capillaries. Volume rendering procedures can then be used to generate a 3D model. However, the preparation of such slices leads to misalignments in relative slice orientation between consecutive slices. Thus, image registration algorithms are necessary to re-align the slices. Here, we present an algorithm for the registration and reconstruction of a vascular network from histologic slices applied to 169 tumor slices. The registration includes two steps. First, consecutive images are incrementally pre-aligned using feature- and area-based transformations. Second, using the previous transformations, parallel registration for all images is enabled. Combining intensity- and color-based thresholds along with heuristic analysis, vascular structures are segmented. A 3D interpolation technique is used for volume rendering. This results in a 3D vascular network with approximately 400–450 vessels with diameters down to 25–30 µm. A delineation of vessel structures with close distance was limited in areas of high structural density. Improvement can be achieved by using images with higher resolution and or machine learning techniques. Full article

Proper functioning of the neural retina relies on the unique retinal environment regulated by the blood–retinal barrier (BRB), which restricts the passage of solutes, fluids, and toxic substances. BRB impairment occurs in many retinal vascular diseases and the breakdown of BRB significantly contributes to disease pathology. Understanding the different molecular constituents and signaling pathways involved in BRB development and maintenance is therefore crucial in developing treatment modalities. This review summarizes the major molecular signaling pathways involved in inner BRB (iBRB) formation and maintenance, and representative animal models of eye diseases with retinal vascular leakage. Studies on Wnt/β-catenin signaling are highlighted, which is critical for retinal and brain vascular angiogenesis and barriergenesis. Moreover, multiple in vivo and in vitro methods for the detection and analysis of vascular leakage are described, along with their advantages and limitations. These pre-clinical animal models and methods for assessing iBRB provide valuable experimental tools in delineating the molecular mechanisms of retinal vascular diseases and evaluating therapeutic drugs. Full article

The nasal mucosa, being accessible and highly vascularized, opens up new opportunities for the systemic administration of drugs. However, there are several protective functions like the mucociliary clearance, a physiological barrier which represents is a difficult obstacle for drug candidates to overcome. For this reason, effective testing procedures are required in the preclinical phase of pharmaceutical development. Based on a recently reported immortalized porcine nasal epithelial cell line, we developed a test platform based on a tissue-compatible microfluidic chip. In this study, a biomimetic glass chip, which was equipped with a controlled bidirectional airflow to induce a physiologically relevant wall shear stress on the epithelial cell layer, was microfabricated. By developing a membrane transfer technique, the epithelial cell layer could be pre-cultivated in a static holder prior to cultivation in a microfluidic environment. The dynamic cultivation within the chip showed a homogenous distribution of the mucus film on top of the cell layer and a significant increase in cilia formation compared to the static cultivation condition. In addition, the recording of the ciliary transport mechanism by microparticle image velocimetry was successful. Using FITC-dextran 4000 as an example, it was shown that this nasal mucosa on a chip is suitable for permeation studies. The obtained permeation coefficient was in the range of values determined by means of other established in vitro and in vivo models. This novel nasal mucosa on chip could, in future, be automated and used as a substitute for animal testing. Full article

Innovative strategies have shown beneficial effects in healing wound management involving, however, a time-consuming and arduous process in clinical contexts. Micro-fragmented skin tissue acts as a slow-released natural scaffold and continuously delivers growth factors, and much other modulatory information, into the microenvironment surrounding damaged wounds by a paracrine function on the resident cells which supports the regenerative process. In this study, in vitro and in vivo investigations were conducted to ascertain improved effectiveness and velocity of the wound healing process with the application of fragmented dermo-epidermal units (FdeU), acquired via a novel medical device (Hy-Tissue^® Micrograft Technology). MTT test; LDH test; ELISA for growth factor investigation (IL) IL-2, IL-6, IL-7 IL-8, IL-10; IGF-1; adiponectin; Fibroblast Growth Factor (FGF); Vascular Endothelial Growth Factor (VEGF); and Tumor Necrosis Factor (TNF) were assessed. Therefore, clinical evaluation in 11 patients affected by Chronic Wounds (CW) and treated with FdeU were investigated. Functional outcome was assessed pre-operatory, 2 months after treatment (T0), and 6 months after treatment (T1) using the Wound Bed Score (WBS) and Vancouver Scar Scale (VSS). In this current study, we demonstrate the potential of resident cells to proliferate from the clusters of FdeU seeded in a monolayer that efficiently propagate the chronic wound. Furthermore, in this study we report how the discharge of trophic/reparative proteins are able to mediate the in vitro paracrine function of proliferation, migration, and contraction rate in fibroblasts and keratinocytes. Our investigations recommend FdeU as a favorable tool in wound healing, displaying in vitro growth-promoting potential to enhance current therapeutic mechanisms. Full article