Search Results (31)

Background/Objectives: The renin–angiotensin system (RAS) is well-established as a moderator of cardiovascular equilibrium and blood pressure. Nevertheless, growing evidence indicates that angiotensin II (Ang II), the principal RAS effector peptide, together with additional constituents, is involved in various malignancies. Since the immune system is an important aspect in tumor development, this study sought to investigate the role of Ang II in the crosstalk between tumor-associated macrophages (TAMs) and breast cancer cells in the tumor microenvironment (TME). Methods: We treated THP-1-like macrophages with 100 nM Ang II for 24 h. The culture media thus obtained was used as conditioned media and applied at 50% on MCF-7 and MDA-MB-231 breast cancer cell lines. The effects of the conditioned media on cancer cell lines were then investigated by various methods such as a cell proliferation assay, migration assay, polarization assay, and by the detection of apoptosis and reactive oxygen species (ROS) generation. Results: We demonstrated that in vitro Ang II promotes macrophage polarization toward proinflammatory M1-like macrophages and anti-inflammatory M2-like macrophages. Interestingly, Ang II, through macrophages, showed varied effects on different breast cancer cell lines, promoting tumor growth and progression in MCF-7 while inhibiting tumor growth and progression in MDA-MB-23. Conclusions: This study has provided clear evidence that Ang II in the TME modulates TAM polarization and secretions, leading to different effects based on the type of breast cancer. Full article

Secreted protein acidic rich in cysteine (SPARC), one of the extracellular matrix proteins, is highly induced during inflammation. We investigated the pathophysiological regulation and role of SPARC in vascular inflammation in a rat model of hypertension created using deoxycorticosterone acetate (DOCA, 40 mg/kg/week, s.c.) and salt (1% in drinking water). DOCA–salt administration time-dependently increased systolic blood pressure during the 3-week treatment period, blunted endothelium-dependent vasodilation, and increased monocyte chemoattractant protein-1 (MCP-1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression in the aorta. SPARC expression transiently increased until week 2 in the DOCA–salt rat aorta. Interestingly, aortic SPARC levels correlated with blood pressure and the levels of MCP-1 and LOX-1 during 0–2 weeks. The AT₁ receptor blocker, losartan, suppressed the overexpression of SPARC, and in vitro treatment with angiotensin II enhanced the production of SPARC in rat aortic endothelial cells. Exposure to recombinant SPARC protein induced overexpression of MCP-1 and LOX-1 mRNA in endothelial cells. Bioactive forms of a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1), excessive activation of which contributes to pathological states and overexpression of which is reported to be induced by SPARC, were increased in the DOCA–salt rat aorta. These results suggest that SPARC is induced by the vascular renin–angiotensin system and causes inflammation in the early stages of hypertensive vascular injury, and that activation of ADAMTS1 might be related to the proinflammatory effects of SPARC. Full article

The renin–angiotensin system helps regulate the endocrine system in modulating blood pressure, fluid volume, and body fluid electrolyte levels. The disruption of the renin–angiotensin system can lead to kidney disease onset and progression. However, the mechanism by which kidney angiotensinogen expression and secretion induce the onset and progression of diabetic nephropathy remains unclear. In this study, we used renal proximal tubular epithelial cells, which express high levels of angiotensinogen, to examine food components that regulate angiotensinogen secretion. The renal proximal tubular epithelial cells were first treated with catalase (antioxidant), daidzein, equol (an isoflavone), a MAP kinase inhibitor, ERK, p38, or JNK and then stimulated with hydrogen peroxide. After 24 h, we collected a culture medium to perform an enzyme-linked immunosorbent assay test for angiotensinogen and cells in order to perform real-time PCR to detect angiotensinogen. We found that angiotensinogen secretion increased as the hydrogen peroxide concentration increased. Catalase, daidzein, and equol decreased angiotensinogen expression and secretion. To investigate the cell signaling mechanism involved in these effects, we assessed the contribution of the MAP kinase cascade. Our data suggest the contribution of p38 and JNK. Our study shows that, in proximal tubular epithelial cells, hydrogen peroxide stimulates angiotensinogen secretion. Isoflavones and p38 inhibited angiotensinogen secretion. Full article

Acute myocardial infarction (AMI) is one of the leading causes of cardiovascular death worldwide. AMI with cardiomyopathy is accompanied by a poor long-term prognosis. However, limited studies have focused on the mechanism of cardiomyopathy associated with AMI. Pericytes are important to the microvascular function in the heart, yet little attention has been paid to their function in myocardial infarction until now. In this study, we integrated single-cell data from individuals with cardiomyopathy and myocardial infarction (MI) GWAS data to reveal the potential function of pericytes in cardiomyopathy-associated MI. We found that pericytes were concentrated in the left atrium and left ventricle tissues. DLC1/GUCY1A2/EGFLAM were the top three uniquely expressed genes in pericytes (p < 0.05). The marker genes of pericytes were enriched in renin secretion, vascular smooth muscle contraction, gap junction, purine metabolism, and diabetic cardiomyopathy pathways (p < 0.05). Among these pathways, the renin secretion and purine metabolism pathways were also found in the process of MI. In cardiomyopathy patients, the biosynthesis of collagen, modulating enzymes, and collagen formation were uniquely negatively regulated in pericytes compared to other cell types (p < 0.05). COL4A2/COL4A1/SMAD3 were the hub genes in pericyte function involved in cardiomyopathy and AMI. In conclusion, this study provides new evidence about the importance of pericytes in the pathogenesis of cardiomyopathy-associated MI. DLC1/GUCY1A2/EGFLAM were highly expressed in pericytes. The hub genes COL4A2/COL4A1/SMAD3 may be potential research targets for cardiomyopathy-associated MI. Full article

The tumor microenvironment comprises multiple cell types, like cancer cells, endothelial cells, fibroblasts, and immune cells. In recent years, there have been massive research efforts focusing not only on cancer cells, but also on other cell types of the tumor microenvironment, thereby aiming to expand and determine novel treatment options. Fibroblasts represent a heterogenous cell family consisting of numerous subtypes, which can alter immune cell fractions, facilitate or inhibit tumor growth, build pre-metastatic niches, or stabilize vessels. These effects can be achieved through cell–cell interactions, which form the extracellular matrix, or via the secretion of cytokines or chemokines. The pro- or antitumorigenic fibroblast phenotypes show variability not only among different cancer entities, but also among intraindividual sites, including primary tumors or metastatic lesions. Commonly prescribed for arterial hypertension, the inhibitors of the renin–angiotensin system have recently been described as having an inhibitory effect on fibroblasts. This inhibition leads to modified immune cell fractions and increased tissue stiffness, thereby contributing to overcoming therapy resistance and ultimately inhibiting tumor growth. However, it is important to note that the inhibition of fibroblasts can also have the opposite effect, potentially resulting in increased tumor growth. We aim to summarize the latest state of research regarding fibroblast heterogeneity and its intricate impact on the tumor microenvironment and extracellular matrix. Specifically, we focus on highlighting recent advancements in the comprehension of intraindividual heterogeneity and therapy options within this context. Full article

Podocytes are highly specialized cells that play a pivotal role in the blood filtration process in the glomeruli of the kidney, and their dysfunction leads to renal diseases. For this reason, the study and application of this cell type is of great importance in the field of regenerative medicine. Hypertension is mainly regulated by the renin–angiotensin–aldosterone system (RAAS), with its main mediator being angiotensin II (ANG II). Elevated ANG II levels lead to a pro-fibrotic, inflammatory, and hypertrophic milieu that induces apoptosis in podocytes. The activation of RAAS is critical for the pathogenesis of podocyte injury; as such, to prevent podocyte damage, patients with hypertension are administered drugs that modulate RAAS signaling. A prime example is the orally active, non-peptide, selective angiotensin-II-type I receptor (AGTR1) blocker losartan. Here, we demonstrate that SIX2-positive urine-derived renal progenitor cells (UdRPCs) and their immortalized counterpart (UM51-hTERT) can be directly differentiated into mature podocytes. These podocytes show activation of RAAS after stimulation with ANG II, resulting in ANG II-dependent upregulation of the expression of the angiotensin-II-type I receptor, AGTR1, and the downregulated expression of the angiotensin-II-type II receptor 2 (AGTR2). The stimulation of podocytes with losartan counteracts ANG II-dependent changes, resulting in a dependent favoring of the specific receptor from AGTR1 to AGTR2. Transcriptome analysis revealed 94 losartan-induced genes associated with diverse biological processes and pathways such as vascular smooth muscle contraction, the oxytocin signaling pathway, renin secretion, and ECM-receptor interaction. Co-stimulation with losartan and ANG II induced the exclusive expression of 106 genes associated with DNA methylation or demethylation, cell differentiation, the developmental process, response to muscle stretch, and calcium ion transmembrane transport. These findings highlight the usefulness of UdRPC-derived podocytes in studying the RAAS pathway and nephrotoxicity in various kidney diseases. Full article

Numerous proteomic and transcriptomic studies have been carried out to better understand the current multi-variant SARS-CoV-2 virus mechanisms of action and effects. However, they are mostly centered on mRNAs and proteins. The effect of the virus on human post-transcriptional regulatory agents such as microRNAs (miRNAs), which are involved in the regulation of 60% of human gene activity, remains poorly explored. Similar to research we have previously undertaken with other viruses such as Ebola and HIV, in this study we investigated the miRNA profile of lung epithelial cells following infection with SARS-CoV-2. At the 24 and 72 h post-infection time points, SARS-CoV-2 did not drastically alter the miRNome. About 90% of the miRNAs remained non-differentially expressed. The results revealed that miR-1246, miR-1290 and miR-4728-5p were the most upregulated over time. miR-196b-5p and miR-196a-5p were the most downregulated at 24 h, whereas at 72 h, miR-3924, miR-30e-5p and miR-145-3p showed the highest level of downregulation. In the top significantly enriched KEGG pathways of genes targeted by differentially expressed miRNAs we found, among others, MAPK, RAS, P13K-Akt and renin secretion signaling pathways. Using RT-qPCR, we also showed that SARS-CoV-2 may regulate several predicted host mRNA targets involved in the entry of the virus into host cells (ACE2, TMPRSS2, ADAM17, FURIN), renin–angiotensin system (RAS) (Renin, Angiotensinogen, ACE), innate immune response (IL-6, IFN1β, CXCL10, SOCS4) and fundamental cellular processes (AKT, NOTCH, WNT). Finally, we demonstrated by dual-luciferase assay a direct interaction between miR-1246 and ACE-2 mRNA. This study highlights the modulatory role of miRNAs in the pathogenesis of SARS-CoV-2. Full article

(1) Background: Renal dysfunction and hypertension are mutually aggravating factors; however, the details of their interaction remain unclear. In a study using renal tissue from diabetic rats, we found that β1-integrin, a cell-substrate adhesion molecule, is specifically phosphorylated in juxtaglomerular cells that secrete renin, a blood pressure regulator. (2) Methods: A mouse juxtaglomerular cell line (As4.1 cells) was used for the following experiments: drug-induced promotion of β1-integrin phosphorylation/dephosphorylation; knockdown of β1-integrin and the cell adhesion molecule connexin-40 (a candidate for the main body of baroreceptor); and pressurization to atmospheric pressure + 100 mmHg. culture in hypotonic liquid medium. The expression of renin under these conditions was measured by qRT-PCR. (3) Results: Phosphorylation of β1-integrin suppressed the expression of renin, while dephosphorylation conversely promoted it. β1-integrin and connexin-40 knockdown both promoted the expression of renin. Pneumatic pressurization and hypotonic medium culture both decreased the expression of renin, which was restored by the knockdown of β1-integrin. (4) Conclusions: β1-integrin plays an inhibitory role in the regulation of the expression of renin, which may be controlled by phosphorylation and dephosphorylation. It is hypothesized that β1-integrin and other adhesion factors regulate the expression of renin by altering the sensitivity of baroreceptors on the plasma membrane. Full article

Obesity, along with type 2 diabetes mellitus (T2DM), is a major contributor to hypertension. The renin-angiotensin-aldosterone system is involved in the occurrence of diabetes and hypertension. However, the mechanism by which obesity is related to T2DM induced hypertension is unclear. In this study, we observed that blood pressure and serum renin content were increased in patients with diabetes and hypertension. Hydrogen sulfide (H₂S), as an endogenous bioactive molecule, has been shown to be a vasodilator. Db/db mice, characterized by obesity and T2DM, and juxtaglomerular (JG) cells, which line the afferent arterioles at the entrance of the glomeruli to produce renin, treated with glucose, palmitic acid (PA) and oleic acid (OA), were used as animal and cellular models. NaHS, the H₂S donor, was administered to db/db mice through intraperitoneal injection. NaHS significantly alleviated blood pressure in db/db mice, decreased the renin content in the serum of db/db mice and reduced renin secretion from JG cells. NaHS modulated renin release via cAMP and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2), which mediate renin exocytosis. Furthermore, NaHS increased the levels of autophagy-related proteins and colocalization with EGFP-LC3 puncta with renin-containing granules and VAMP2 to consume excessive renin to maintain intracellular homeostasis. Therefore, exogenous H₂S attenuates renin release and promotes renin-vesicular autophagy to relieve diabetes-induced hypertension. Full article

The seminal discoveries that parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) are major endocrine regulators of vitamin D metabolism led to a significant improvement in our understanding of the pivotal roles of peptide hormones and small proteohormones in the crosstalk between different organs, regulating vitamin D metabolism. The interaction of vitamin D, FGF23 and PTH in the kidney is essential for maintaining mineral homeostasis. The proteohormone FGF23 is mainly secreted from osteoblasts and osteoclasts in the bone. FGF23 acts on proximal renal tubules to decrease production of the active form of vitamin D (1,25(OH)₂D) by downregulating transcription of 1α-hydroxylase (CYP27B1), and by activating transcription of the key enzyme responsible for vitamin D degradation, 24-hydroxylase (CYP24A1). Conversely, the peptide hormone PTH stimulates 1,25(OH)₂D renal production by upregulating the expression of 1α-hydroxylase and downregulating that of 24-hydroxylase. The circulating concentration of 1,25(OH)₂D is a positive regulator of FGF23 secretion in the bone, and a negative regulator of PTH secretion from the parathyroid gland, forming feedback loops between kidney and bone, and between kidney and parathyroid gland, respectively. In recent years, it has become clear that vitamin D signaling has important functions beyond mineral metabolism. Observation of seasonal variations in blood pressure and the subsequent identification of vitamin D receptor (VDR) and 1α-hydroxylase in non-renal tissues such as cardiomyocytes, endothelial and smooth muscle cells, suggested that vitamin D may play a role in maintaining cardiovascular health. Indeed, observational studies in humans have found an association between vitamin D deficiency and hypertension, left ventricular hypertrophy and heart failure, and experimental studies provided strong evidence for a role of vitamin D signaling in the regulation of cardiovascular function. One of the proposed mechanisms of action of vitamin D is that it functions as a negative regulator of the renin-angiotensin-aldosterone system (RAAS). This finding established a novel link between vitamin D and RAAS that was unexplored until then. During recent years, major progress has been made towards a more complete understanding of the mechanisms by which FGF23, PTH, and RAAS regulate vitamin D metabolism, especially at the genomic level. However, there are still major gaps in our knowledge that need to be filled by future research. The purpose of this review is to highlight our current understanding of the molecular mechanisms underlying the interaction between vitamin D, FGF23, PTH, and RAAS, and to discuss the role of these mechanisms in physiology and pathophysiology. Full article

In this review, we describe previous basic and clinical studies on autonomous aldosterone production. Over the past decades, mineralocorticoid receptor antagonists (MRAs) have been found to concentration-dependently inhibit steroidogenesis in different degrees. However, many studies have proven the suppressive effects of MRAs on the activities of hormone synthase. The probable factors of cytochrome P-450 reduction, both in microsomes and mitochondria, have also been considered: (1) one of the spironolactone metabolite forms had destructive function, except canrenone, (2) 7α-thio-spironolactone was an obligatory intermediate in the spironolactone-induced CYP450 decrease, and (3) the contributing steroids should have 7α-methylthio or 7α-methylsulfone groups. In previous clinical research, spironolactone-body-containing cells showed a type II pattern of enzyme activity (i.e., enhanced 3β-hydroxysteroid dehydrogenase, glucose-6-phosphate, and NADP-isocitrate dehydrogenase activities and weaken succinate dehydrogenase activity), and the subcapsular micronodules composed of spironolactone-body-containing cells also exhibited a type II pattern and excess aldosterone secretion, indicating that the subcapsular micronodules might be the root of aldosterone-producing adenoma. Moreover, combined with the potential impeditive function to aldosterone secretion, a few cases of spontaneous remission of primary aldosteronism, with normal ranges of blood pressure, plasma potassium, plasma renin activity, and aldosterone renin ratio, have been reported after long-term treatment with MRAs. Full article

Low back pain is a clinically highly relevant musculoskeletal burden and is associated with inflammatory as well as degenerative processes of the intervertebral disc. However, the pathophysiology and cellular pathways contributing to this devastating condition are still poorly understood. Based on previous evidence, we hypothesize that tissue renin-angiotensin system (tRAS) components, including the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2), are present in human nucleus pulposus (NP) cells and associated with inflammatory and degenerative processes. Experiments were performed with NP cells from four human donors. The existence of angiotensin II, angiotensin II type 1 receptor (AGTR1), AGTR2, MAS-receptor (MasR), and ACE2 in human NP cells was validated with immunofluorescent staining and gene expression analysis. Hereafter, the cell viability was assessed after adding agonists and antagonists of the target receptors as well as angiotensin II in different concentrations for up to 48 h of exposure. A TNF-α-induced inflammatory in vitro model was employed to assess the impact of angiotensin II addition and the stimulation or inhibition of the tRAS receptors on inflammation, tissue remodeling, expression of tRAS markers, and the release of nitric oxide (NO) into the medium. Furthermore, protein levels of IL-6, IL-8, IL-10, and intracellular as well as secreted angiotensin II were assessed after exposing the cells to the substances, and inducible nitric oxide synthase (iNOS) levels were evaluated by utilizing Western blot. The existence of tRAS receptors and angiotensin II were validated in human NP cells. The addition of angiotensin II only showed a mild impact on gene expression markers. However, there was a significant increase in NO secreted by the cells. The gene expression ratios of pro-inflammatory/anti-inflammatory cytokines IL-6/IL-10, IL-8/IL-10, and TNF-α/IL-10 were positively correlated with the AGTR1/AGTR2 and AGTR1/MAS1 ratios, respectively. The stimulation of the AGTR2 MAS-receptor and the inhibition of the AGTR1 receptor revealed beneficial effects on the gene expression of inflammatory and tissue remodeling markers. This finding was also present at the protein level. The current data showed that tRAS components are expressed in human NP cells and are associated with inflammatory and degenerative processes. Further characterization of the associated pathways is warranted. The findings indicate that tRAS modulation might be a novel therapeutic approach to intervertebral disc disease. Full article

Dyslipidemia is described as a hallmark of metabolic syndrome, promoting a stage of metabolic inflammation (metainflammation) that could lead to misbalances in energetic metabolism, contributing to insulin resistance, and modifying intracellular cholesterol pathways and the renin–angiotensin system (RAS) in pancreatic islets. Low-density lipoprotein (LDL) hypercholesterolemia could disrupt the tissue communication between Langerhans β-cells and hepatocytes, wherein extracellular vesicles (EVs) are secreted by β-cells, and exposition to LDL can impair these phenomena. β-cells activate compensatory mechanisms to maintain insulin and metabolic homeostasis; therefore, the work aimed to characterize the impact of LDL on β-cell cholesterol metabolism and the implication on insulin secretion, connected with the regulation of cellular communication mediated by EVs on hepatocytes. Our results suggest that β-cells can endocytose LDL, promoting an increase in de novo cholesterol synthesis targets. Notably, LDL treatment increased mRNA levels and insulin secretion; this hyperinsulinism condition was associated with the transcription factor PDX-1. However, a compensatory response that maintains basal levels of intracellular calcium was described, mediated by the overexpression of calcium targets PMCA1/4, SERCA2, and NCX1, together with the upregulation of the unfolded protein response (UPR) through the activation of IRE1 and PERK arms to maintain protein homeostasis. The LDL treatment induced metainflammation by IL-6, NF-κB, and COX-2 overexpression. Furthermore, LDL endocytosis triggered an imbalance of the RAS components. LDL treatment increased the intracellular levels of cholesterol on lipid droplets; the adaptive β-cell response was portrayed by the overexpression of cholesterol transporters ABCA1 and ABCG1. Therefore, lipotoxicity and hyperinsulinism induced by LDL were regulated by the natural compound auraptene, a geranyloxyn coumarin modulator of cholesterol-esterification by ACAT1 enzyme inhibition. EVs isolated from β-cells impaired insulin signaling via mTOR/p70S6Kα in hepatocytes, a phenomenon regulated by auraptene. Our results show that LDL overload plays a novel role in hyperinsulinism, mechanisms associated with a dysregulation of intracellular cholesterol, lipotoxicity, and the adaptive UPR, which may be regulated by coumarin-auraptene; these conditions explain the affectations that occur during the initial stages of insulin resistance. Full article

Lipopolysaccharide (LPS) is associated with chronic intestinal inflammation and promotes intestinal cancer progression in the gut. While the interplay between LPS and intestinal immune cells has been well-characterized, little is known about LPS and the intestinal epithelium interactions. In this study, we explored the differential effects of LPS on proliferation and the transcriptome in 3D enteroids/colonoids obtained from dogs with naturally occurring gastrointestinal (GI) diseases including inflammatory bowel disease (IBD) and intestinal mast cell tumor. The study objective was to analyze the LPS-induced modulation of signaling pathways involving the intestinal epithelia and contributing to colorectal cancer development in the context of an inflammatory (IBD) or a tumor microenvironment. While LPS incubation resulted in a pro-cancer gene expression pattern and stimulated proliferation of IBD enteroids and colonoids, downregulation of several cancer-associated genes such as Gpatch4, SLC7A1, ATP13A2, and TEX45 was also observed in tumor enteroids. Genes participating in porphyrin metabolism (CP), nucleocytoplasmic transport (EEF1A1), arachidonic acid, and glutathione metabolism (GPX1) exhibited a similar pattern of altered expression between IBD enteroids and IBD colonoids following LPS stimulation. In contrast, genes involved in anion transport, transcription and translation, apoptotic processes, and regulation of adaptive immune responses showed the opposite expression patterns between IBD enteroids and colonoids following LPS treatment. In brief, the crosstalk between LPS/TLR4 signal transduction pathway and several metabolic pathways such as primary bile acid biosynthesis and secretion, peroxisome, renin–angiotensin system, glutathione metabolism, and arachidonic acid pathways may be important in driving chronic intestinal inflammation and intestinal carcinogenesis. Full article

Preeclampsia (PE) is a pregnancy-specific disorder that affects 3 to 5% of pregnancies worldwide and is one of the leading causes of maternal and fetal morbidity and mortality. Nevertheless, how these events occur remains unclear. We hypothesized that the induction of hypoxic conditions in vitro in primary human trophoblast cells would mimic several characteristics of PE found in vivo. We applied and characterized a model of primary cytotrophoblasts isolated from healthy pregnancies that were placed under different oxygen concentrations: ambient O₂ (5% pCO₂, 21%pO₂, 24 h, termed “normoxia”), low O₂ concentration (5% pCO₂, 1.5% pO₂, 24 h, termed “hypoxia”), or “hypoxia/reoxygenation” (H/R: 6 h intervals of normoxia and hypoxia for 24 h). Various established preeclamptic markers were assessed in this cell model and compared to placental tissues obtained from PE pregnancies. Seventeen PE markers were analyzed by qPCR, and the protein secretion of soluble fms-like tyrosine kinase 1 (sFlT-1) and the placenta growth factor (PlGF) was determined by ELISA. Thirteen of seventeen genes associated with angiogenesis, the renin–angiotensin system, oxidative stress, endoplasmic reticulum stress, and the inflammasome complex were susceptible to H/R and hypoxia, mimicking the expression pattern of PE tissue. In cell culture supernatants, the secretion of sFlT-1 was increased in hypoxia, while PlGF release was significantly reduced in H/R and hypoxia. In the supernatants of our cell models, the sFlT-1/PlGF ratio in hypoxia and H/R was higher than 38, which is a strong indicator for PE in clinical practice. These results suggest that our cellular models reflect important pathological processes occurring in PE and are therefore suitable as PE in vitro models. Full article