Type V Collagen in Scar Tissue Regulates the Size of Scar after Heart Injury.

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.

Integrated analyses reveal the diagnostic and predictive values of COL5A2 and association with immune environment in Crohn's disease.

The pathogenesis of Crohn's disease (CD) involves abnormal immune cell infiltration and dysregulated immune response. Therefore, thorough research on immune cell abnormalities in CD is crucial for improved treatment of this disease. Single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data of CD were obtained from the Gene Expression Omnibus (GEO) database. Cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT), weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) networks evaluated the proportion of immune infiltrating cells, constructed co-expression network and identified key genes, respectively. Based on the dataset (GSE134809), 15 cell clusters were defined and labeled as different cell types. Among the 11 modules, the yellow module had the closest relationship with plasma cells (cluster 5). Confirmed using RNA sequencing and IHC assay, the expression of COL5A2 in CD samples was higher than that in control samples. Furthermore, the COL5A2 protein expression remarkably decreased in the group of patients who responded to anti-tumor necrosis factor (TNF) treatments, compared to the non-response group. The comprehensive analyses described here provided novel insight into the landscape of CD-associated immune environment. In addition, COL5A2 were identified as potential diagnostic indicators for CD, as well as promising predictive markers for CD patients.

Molecular alterations due to Col5a1 haploinsufficiency in a mouse model of classic Ehlers-Danlos syndrome.

Type V collagen is a regulatory fibrillar collagen essential for type I collagen fibril nucleation and organization and its deficiency leads to structurally abnormal extracellular matrix (ECM). Haploinsufficiency of the Col5a1 gene encoding α(1) chain of type V collagen is the primary cause of classic Ehlers-Danlos syndrome (EDS). The mechanisms by which this initial insult leads to the spectrum of clinical presentation are not fully understood. Using transcriptome analysis of skin and Achilles tendons from Col5a1 haploinsufficient (Col5a1+/-) mice, we recognized molecular alterations associated with the tissue phenotypes. We identified dysregulation of ECM components including thrombospondin-1, lysyl oxidase, and lumican in the skin of Col5a1+/- mice when compared with control. We also identified upregulation of transforming growth factor ß1 (Tgf-ß) in serum and increased expression of pSmad2 in skin from Col5a1+/- mice, suggesting Tgf-ß dysregulation is a contributor to abnormal wound healing and atrophic scarring seen in classic EDS. Together, these findings support altered matrix to cell signaling as a component of the pathogenesis of the tissue phenotype in classic EDS and point out potential downstream signaling pathways that may be targeted for the treatment of this disease.

A novel cancer-associated fibroblasts risk score model predict survival and immunotherapy in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related death worldwide. Cancer-associated fibroblasts (CAFs) are a special type of fibroblasts, which play an important role in the development and immune escape of tumors. Weighted gene co-expression network analysis (WGCNA) was used to construct the co-expression module. In combination with univariate Cox regression and analysis of least absolute shrinkage operator (LASSO), characteristics associated with CAFs were developed for a prognostic model. The migration and proliferation of lung cancer cells were evaluated in vitro. Finally, the expression levels of proteins were analyzed by Western blot. LASSO Cox regression algorithm was then performed to select hub genes. Finally, a total of 2 Genes (COL5A2, COL6A2) were obtained. We then divided LUAD patients into high- and low-risk groups based on CAFs risk scores. Survival analysis, CAFs score correlation analysis and tumor mutation load analysis showed that COL5A2 and COL6A2 were high-risk genes for LUAD. Human Protein Atlas (HPA), western blot and PCR results showed that COL5A2 and COL6A2 were up-regulated in LUAD tissues. When COL5A2 and COL6A2 were knocked down, the proliferation, invasion and migration of lung cancer cells were significantly decreased. Finally, COL5A2 can affect LUAD progression through the Wnt/ß-Catenin and TGF-ß signaling pathways. Our CAFs risk score model offers a new approach for predicting the prognosis of LUAD patients. Furthermore, the identification of high-risk genes COL5A2 and COL6A2 and drug sensitivity analysis can provide valuable candidate clues for clinical treatment of LUAD.

The Association of a Single Nucleotide Variant in COL5A1 to Early Onset Keratoconus and Pectus Excavatum-Convergence of Extracellular Matrix Pathologies.

Keratoconus is a bilateral ocular condition characterized by irregularities and the thinning of the cornea. Decreased central corneal thickness is a hallmark of the condition, and numerous genes have played a role in altering corneal thickness and the subsequent development of keratoconus. Variants in the structural and regulatory genes of the extracellular matrix have been highly associated with keratoconus, as well as with pectus excavatum, a chest wall deformity commonly seen in connective tissue disorders. This report describes a patient with a c.1720-11T>A intronic variant in the collagen-encoding gene, COL5A1, who was diagnosed with early-onset keratoconus and demonstrated a significant pectus excavatum. This report associates a COL5A1 variant with these seemingly unrelated phenotypic associations, further advancing the literature on the topic.

LINC00173 blocks GATA6-mediated transcription of COL5A1 to affect malignant development of oral squamous cell carcinoma.

BACKGROUND: Aberrant expression of collagen type V alpha 1 chain (COL5A1) has been linked to several forms of human cancers. In this work, we focused on the interaction of the LINC00173/GATA binding protein 6 (GATA6)/COL5A1 axis in the malignant property of oral squamous cell carcinoma (OSCC) cells. METHODS: We analyzed six publicly accessible datasets GSE160042, GSE74530, GSE138206, GSE23558, GSE31853 and GSE146483 to identify aberrantly expressed genes in OSCC. The expression of COL5A1 in OSCC tissues and cell lines was examined by reverse transcription-quantitative polymerase chain reaction and/or immunohistochemistry. The regulatory mechanism responsible for COL5A1 transcription was predicted via bioinformatics systems, and the interactions of LINC00173, GATA6, and COL5A1 were identified by immunoprecipitation and luciferase assays. Overexpression or downregulation of COL5A1, GATA6, and LINC00173 were induced in OSCC cell lines to determine their roles in the malignant phenotype of the OSCC cells in vitro and in vivo. RESULTS: COL5A1 showed elevated expression in OSCC tissues and cells. The COLA51 knockdown suppressed proliferation, migration and invasiveness, apoptosis resistance, and pro-angiogenic ability of OSCC cells, and it suppressed the growth and dissemination of xenograft tumors in vivo. GATA6 bound to COL5A1 promoter to activate its transcription, whereas LINC00173 bound to GATA6 to block this transcriptional activation. Overexpression of GATA6 or COL5A1 promoted the malignant phenotype of the OSCC cells, which were blocked upon LINC00173 upregulation. CONCLUSION: This work demonstrates that LINC00173 blocks GATA6-mediated transcription of COL5A1 to affect malignant development of OSCC.

LncRNA BBOX1-AS1 Contributes to the Progression of Esophageal Carcinoma by Targeting the miR-361-3p/COL5A1 Axis.

Long noncoding RNAs (lncRNAs) are known to participate in the progression of several cancers, including esophageal carcinoma (EC), a common malignancy of the digestive system. Although the role of the lncRNA-miRNA-mRNA regulatory network is crucial for the growth and progression of EC, the regulation of lncRNA BBOX1-AS1 (BBOX1 antisense RNA1) remains unclear. We performed reverse transcription-quantitative PCR (RT-qPCR) and western blotting to evaluate miR-361-3p, collagen type V alpha 1 chain (COL5A1), and BBOX1-AS1 expression levels in EC cells and tissues. The colony formation assay (CFA) and Cell Counting Kit-8 (CCK-8) were employed to identify EC cell proliferation, while western blotting was used to examine EC cell apoptosis and Bax and Bcl-2 expression levels. The effect of BBOX1-AS1 on EC proliferation was determined using an in vivo carcinogenesis assay. Correlation between COL5A1, BBOX1-AS1, and miR-361-3p was examined using the luciferase reporter system and RNA immunoprecipitation assay (RIP). Herein, we observed that BBOX1-AS1 expression levels were upregulated in EC cells and tissues. BBOX1-AS1 knockdown inhibited EC cell proliferation and conferred a pro-apoptotic effect. These results indicated a positive interaction between BBOX1-AS1 and miR-361-3p in EC and a negative association with miR-361-3p. COL5A1 was recognized as a downstream miR-361-3p target and was inversely related to miR-361-3p in EC. Therefore, BBOX1-AS1 expression suppressed cell apoptosis and promoted cell proliferation via the downregulation of miR-361-3p and upregulation of COL5A1 expression. Overall, BBOX1-AS1 facilitates EC progression via the miR-361-3p or COL5A1 axis, indicating that BBOX1-AS1 might be a potential therapeutic target for EC therapy.

Collagen type V alpha 2 promotes the development of gastric cancer via M2 macrophage polarization.

Gastric cancer is a type of digestive tract cancer with a high morbidity and mortality, which leads to a major health burden worldwide. More research into the functions of the immune system will improve therapy and survival in gastric cancer patients. We attempted to identify potential biomarkers or targets in gastric cancer via bioinformatical analysis approaches. Three gene expression profile datasets (GSE79973, GSE103236, and GSE118916) of gastric tissue samples were obtained from the Gene Expression Omnibus database. There were 65 overlapping differentially expressed genes (DEGs) identified from three microarrays. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway were carried out for the key functions and pathways enriched in the DEGs. Then, ten hub genes were identified by protein-protein interaction network. In addition, we observed that collagen type V alpha 2 (COL5A2) was linked to gastric cancer prognosis as well as M2 macrophage infiltration. Furthermore, COL5A2 enhanced gastric cancer cell proliferation through the PI3K-AKT signaling pathway and polarized M2 macrophage cells. Therefore, in this study, we found that COL5A2 was associated with the development of gastric cancer which might function as a potential therapeutic target for the disease.

Collagen Gene Polymorphisms Previously Associated with Resistance to Soft-Tissue Injury Are More Common in Competitive Runners Than Nonathletes.

ABSTRACT: Dines, HR, Nixon, J, Lockey, SJ, Herbert, AJ, Kipps, C, Pedlar, CR, Day, SH, Heffernan, SM, Antrobus, MR, Brazier, J, Erskine, RM, Stebbings, GK, Hall, ECR, and Williams, AG. Collagen gene polymorphisms previously associated with resistance to soft-tissue injury are more common in competitive runners than nonathletes. J Strength Cond Res 37(4): 799-805, 2023-Single-nucleotide polymorphisms (SNPs) of collagen genes have been associated with soft-tissue injury and running performance. However, their combined contribution to running performance is unknown. We investigated the association of 2 collagen gene SNPs with athlete status and performance in 1,429 Caucasian subjects, including 597 competitive runners (354 men and 243 women) and 832 nonathletes (490 men and 342 women). Genotyping for COL1A1 rs1800012 (C > A) and COL5A1 rs12722 (C > T) SNPs was performed by a real-time polymerase chain reaction. The numbers of "injury-resistant" alleles from each SNP, based on previous literature (rs1800012 A allele and rs12722 C allele), were combined as an injury-resistance score (RScore, 0-4; higher scores indicate injury resistance). Genotype frequencies, individually and combined as an RScore, were compared between cohorts and investigated for associations with performance using official race times. Runners had 1.34 times greater odds of being rs12722 CC homozygotes than nonathletes (19.7% vs. 15.5%, p = 0.020) with no difference in the rs1800012 genotype distribution ( p = 0.659). Fewer runners had an RScore 0 of (18.5% vs. 24.7%) and more had an RScore of 4 (0.6% vs. 0.3%) than nonathletes ( p < 0.001). Competitive performance was not associated with the COL1A1 genotype ( p = 0.933), COL5A1 genotype ( p = 0.613), or RScore ( p = 0.477). Although not associated directly with running performance among competitive runners, a higher combined frequency of injury-resistant COL1A1 rs1800012 A and COL5A1 rs12722 C alleles in competitive runners than nonathletes suggests these SNPs may be advantageous through a mechanism that supports, but does not directly enhance, running performance.

Type I and type V procollagen triple helix uses different subsets of the molecular ensemble for lysine posttranslational modifications in the rER.

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

A patient with a novel pathogenic variant in COL5A1 exhibiting prominent vascular and cardiac features.

The Ehlers-Danlos Syndromes (EDS) are a group of inherited connective tissue disorders with a worldwide prevalence of 1 in 2500 to 1 in 5000 births irrespective of sex or ethnicity. Fourteen subtypes of Ehlers-Danlos Syndrome (EDS) have been described, each with characteristic phenotypes and associated genes. Pathogenic variants in COL5A1 and COL5A2 cause the classical EDS subtypes. Pathogenic variants in COL3A1 cause vascular EDS. In this case report, we describe a patient with a phenotype resembling that of vascular EDS, caused by a novel pathogenic variant in COL5A1.

Clinical and molecular characteristics of 168 probands and 65 relatives with a clinical presentation of classical Ehlers-Danlos syndrome.

Classical Ehlers-Danlos syndrome (cEDS) is a heritable connective tissue disorder mainly caused by pathogenic variants in COL5A1 or COL5A2, encoding type V collagen. Its diagnosis, based on clinical criteria and molecular confirmation, can be challenging. We report the molecular and clinical characteristics of 168 probands (72 clinically evaluated at our center) and 65 relatives with a clinical presentation of cEDS. Type V collagen defects were found in 145 probands, 121 (83.5%) were located in COL5A1 and 24 (16.5%) in COL5A2. Although 85.6% of molecularly confirmed patients presented the two major clinical criteria (generalized joint hypermobility, hyperextensible skin with atrophic scarring), significant inter- and intrafamilial phenotypic variability was noted. COL5A2 variants often caused a more severe phenotype. Vascular complications were rare in individuals with type V collagen defects (1.4%). Among the 72 probands clinically evaluated in our center, the mutation detection rate was 82.0%. The majority (68.1%) harbored COL5A1/COL5A2 defects. Yet, 13.9% harbored a defect in another gene (COL1A1, PLOD1, TNXB, AEBP1) highlighting important clinical overlap and the need for molecular confirmation of the diagnosis as this has implications regarding follow-up and genetic counseling. Eighteen percent of the 72 probands remained molecularly unexplained and a COL5A1 variant of unknown significance was identified in 6.9%.

Overexpressed COL5A1 is correlated with tumor progression, paclitaxel resistance, and tumor-infiltrating immune cells in ovarian cancer.

Ovarian cancer (OC) remains the leading cause of cancer-related death among gynecological cancers. The present study examined the role of collagen type V alpha 1 (COL5A1) and the characteristics of COL5A1 as an oncogenic protein in OC. The association of COL5A1 with paclitaxel (PTX)-resistance and stemness in OC was also studied and the multidatabase and big data analyses of the prognostic value, coexpression network, genetic alterations, and tumor-infiltrating immune cells of COL5A1 were elucidated. We found that COL5A1 expression was high in OC cells and tissues. Knockdown of COL5A1 inhibited the proliferation and migration of OC cells. Further study also showed that COL5A1 was overexpressed in PTX-resistant OC cells compared to respective PTX-sensitive cells. Additionally, COL5A1 was more enriched in OC stem cell-like cells. Silencing COL5A1 expression decreased the OC cell resistance to PTX and inhibited the ability of OC-spheroid formation. Survival analysis predicted that the elevated COL5A1 expression was associated with a worse survival outcome and correlated to the tumor stage of OC patients. The estimating relative subsets of RNA transcripts (CIBERSORT) algorithm analysis also unveiled the correlation of several tumor-infiltrating immune cells with the expression of COL5A1. Taken together, our data demonstrate that COL5A1 is a biomarker to predict OC progression and PTX-resistance and represents a promising target for OC treatment.

Collagens in primary frozen shoulder: expression of collagen mRNA isoforms in the different phases of the disease.

OBJECTIVES: Primary frozen shoulder (pFS) has three phases that differ in clinical presentation. It is characterized by contracture of the joint capsule. We hypothesized that there is a general upregulation of collagens in pFS, and that this is highest in the first phase of the disease. The aims of this study were to investigate the expression of various collagens and degradation of collagens in patients with primary pFS and relate this to the three phases of the condition. METHODS: From twenty-six patients with pFS and eight control patients with subacromial impingement, biopsies were obtained during shoulder arthroscopy from the middle glenohumeral ligament and the anterior capsule, and mRNA levels for collagens, MMP-2 and -14 and TGF-ß1, - ß2 and -ß3 in the tissue were analysed using real-time PCR. RESULTS: Genes for collagens type I, III, IV, V, VI and XIV, were activated in pFS, and the total mRNA for all collagens was increased (P < 0.05). This upregulation was independent of disease phases in pFS. In addition, MMP-2, MMP-14, TGF-ß1 and TGF-ß3 were upregulated in all phases of the disease. CONCLUSION: There is a general upregulation and an increased degradation of collagens in pFS in all three phases of the disease. This indicates a constantly increased turnover of the fibrotic tissue in the capsule from pFS. The difference in clinical presentation of pFS observed in the three phases of the disease is not primarily a result of variations in collagen production.

Growth, development, and phenotypic spectrum of individuals with deletions of 2q33.1 involving SATB2.

SATB2-Associated syndrome (SAS) is an autosomal dominant, multisystemic, neurodevelopmental disorder due to alterations in SATB2 at 2q33.1. A limited number of individuals with 2q33.1 contiguous deletions encompassing SATB2 (ΔSAS) have been described in the literature. We describe 17 additional individuals with ΔSAS, review the phenotype of 33 previously published individuals with 2q33.1 deletions (n = 50, mean age = 8.5 ± 7.8 years), and provide a comprehensive comparison to individuals with other molecular mechanisms that result in SAS (non-ΔSAS). Individuals in the ΔSAS group were often underweight for age (20/41 = 49%) with a progressive decline in weight (95% CI = -2.3 to -1.1, p < 0.0001) and height (95% CI = -2.3 to -1.0, p < 0.0001) Z-score means from birth to last available measurement. ΔSAS individuals were often noted to have a broad spectrum of facial dysmorphism. A composite image of ΔSAS individuals generated by automated image analysis was distinct as compared to matched controls and non-ΔSAS individuals. We also present additional genotype-phenotype correlations for individuals in the ΔSAS group such as an increased risk for aortic root/ascending aorta dilation and primary pulmonary hypertension for those individuals with contiguous gene deletions that include COL3A1/COL5A2 and BMPR2, respectively. Based on these findings, we provide additional care recommendations for individuals with ΔSAS variants.

Impaired collagen fibril assembly in keloids with enhanced expression of lumican and collagen V.

Keloids are characterized by fibroblast activation and altered architecture of extracellular matrix (ECM). Excessive deposition of ECM molecules and irregular organization of collagen fibers have been observed in keloids. However, the ultrastructural alteration of collagen has not been fully investigated. In this study, the differences in tissue structure, collagen ultrastructure, matrix components, mechanical properties and collagen assembling molecules between keloids and their extra-lesional skins (ELSs) were explored using histology, transmission electron microscope (TEM), qPCR, Western blot, immunohistochemistry and bioinformatics. Histological evaluation showed thinner fibers in keloids with increased contents of collagen III and proteoglycans, which were supported by TEM findings of thinner collagen fibrils and less developed D-band periodicity in keloids than in ELSs (p < 0.05). In addition, total collagen and water contents were significantly increased (p < 0.05) along with richer proteoglycan production in keloids vs ELSs, which also led to increased stiffness and decreased maximal load in keloids compared with ELSs. Mechanism study showed that multiple molecules related to matrix assembly were significantly upregulated in keloids (p < 0.05). In particular, lumican and collagen V showed high degrees in co-expression analysis and their upregulation levels were revealed from microarray data, which were also verified in keloids at both gene and protein levels (p < 0.05). Nevertheless, siRNA knockdown of lumican failed to affect in vitro collagen assembly, but caused upregulated collagen V expression along with the upregulation of focal adhesion kinase, TGF-ß1, TGF-ß3 and PDGF, among which some are known for capable of enhancing collagen V expression. In conclusion, this study demonstrates impaired collagen assembly along with enhanced expression of lumican and collagen V, both are known for interfering with collagen fibril assembly.

Different mRNA expression patterns in keratoglobus and pellucid marginal degeneration keratocytes.

PURPOSE: Alike keratoconus (KC), keratoglobus (KG) and pellucid marginal degeneration (PMD) belong to ectatic corneal diseases. While there are numerous studies on keratoconus pathophysiology, there is no exact knowledge on genetic and pathophysiological background of KG and PMD, so far. It is not yet clarified, whether KG and PMD are independent clinical entities or represent different stages of the same disease. Our purpose was to investigate key parameters concerning collagen synthesis, intracellular LOX expression and inflammation in corneal stromal cells of KG and PMD subjects, in vitro. METHODS: Normal human keratocytes of corneas from the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz and human keratocytes of KG and PMD patients were isolated and cultured as keratocytes. To examine Collagen I and V (Col I, Col V), heat shock protein 47 (Hsp47), Lysyl Oxidase (LOX), nuclear factor kappa B (NF-κB) mRNA and protein expression in all cell types, quantitative PCR and Western blot analysis has been performed. RESULTS: Col5A1 mRNA expression was significantly lower in KG and PMD keratocytes and LOX mRNA expression was significantly higher in KG-keratocytes, compared to controls. Col1A1, Hsp47 and NF-κB mRNA expression and the analyzed protein expressions did not differ from controls, in KG or PMD. CONCLUSIONS: Col5A1 mRNA expression is decreased in KG and PMD and LOX mRNA expression is increased in KG. Therefore, the pathophysiology of KG and PMD differs from KC and these seem to be from KC independent entities. The explanation of the peripheral corneal thinning in KG and PMD must be investigated in further studies.

Collagen remodelling and plasma ascorbic acid levels in patients suspected of inherited bleeding disorders harbouring germline variants in collagen-related genes.

INTRODUCTION: Variants in collagen-related genes COL1A1, COL3A1, COL5A1 and COL5A2 are associated with Ehlers-Danlos syndrome (EDS), a heterogeneous group of connective tissue disorders strongly associated with increased bleeding. Of patients with incompletely explained bleeding diathesis, a relatively high proportion were shown to harbour at least one heterozygous variant of unknown significance (VUS) in one of these genes, the vast majority without meeting the clinical criteria for EDS. AIM: To investigate the functional consequences of the identified variants by assessing the formation and degradation of types I, III and V collagen, in addition to plasma levels of ascorbic acid (AA). METHODS: A total of 31 patients harbouring at least one heterozygous VUS in COL1A1, COL3A1, COL5A1 or COL5A2 and 20 healthy controls were assessed using monoclonal antibodies targeting neo-epitopes specific for collagen formation and degradation. Plasma AA levels were measured in patients using high-performance liquid chromatography. RESULTS: Serum levels of C5 M (degradation of type V collagen) were decreased in patients compared with healthy controls (p = .033). No significant differences were found in biomarkers for remodelling of types I and III collagen. A significant negative correlation between bleeding (ISTH-BAT score) and plasma AA levels was shown (r = -.42; r2  = .17; p = .020). Suboptimal or marginally deficient AA status was found in 8/31 patients (26%). CONCLUSION: Functional investigations of collagen remodelling were not able to identify any clear associations between the identified variants and increased bleeding. The negative correlation between plasma AA levels and ISTH-BAT score motivates further investigations.

Shear Stress-Induced miR-143-3p in Collateral Arteries Contributes to Outward Vessel Growth by Targeting Collagen V-α2.

OBJECTIVE: Arteriogenesis, describing the process of collateral artery growth, is activated by fluid shear stress (FSS). Since this vascular mechanotransduction may involve microRNAs (miRNAs), we investigated the FSS-induced expression of vascular cell miRNAs and their functional impact on collateral artery growth during arteriogenesis. Approach and Results: To this end, rats underwent femoral artery ligation and arteriovenous anastomosis to increase collateral blood flow to maximize FSS and trigger collateral vessel remodeling. Five days after surgery, a miRNA expression profile was obtained from collateral tissue, and upregulation of 4 miRNAs (miR-24-3p, miR-143-3p, miR-146a-5p, and miR-195-5p) was verified by quantitative polymerase chain reaction. Knockdown of miRNAs at the same time of the surgery in an in vivo mouse ligation and recovery model demonstrated that inhibition of miR-143-3p only severely impaired blood flow recovery due to decreased arteriogenesis. In situ hybridization revealed distinct localization of miR-143-3p in the vessel wall of growing collateral arteries predominantly in smooth muscle cells. To investigate the mechanotransduction of FSS leading to the increased miR-143-3p expression, cultured endothelial cells were exposed to FSS. This provoked the expression and release of TGF-ß (transforming growth factor-ß), which increased the expression of miR-143-3p in smooth muscle cells in the presence of SRF (serum response factor) and myocardin. COL5A2 (collagen type V-α2)-a target gene of miR-143-3p predicted by in silico analysis-was found to be downregulated in growing collaterals. CONCLUSIONS: These results indicate that the increased miR-143-3p expression in response to FSS might contribute to the reorganization of the extracellular matrix, which is important for vascular remodeling processes, by inhibiting collagen V-α2 biosynthesis.

A Novel Recurrent COL5A1 Genetic Variant Is Associated With a Dysplasia-Associated Arterial Disease Exhibiting Dissections and Fibromuscular Dysplasia.

OBJECTIVE: While rare variants in the COL5A1 gene have been associated with classical Ehlers-Danlos syndrome and rarely with arterial dissections, recurrent variants in COL5A1 underlying a systemic arteriopathy have not been described. Monogenic forms of multifocal fibromuscular dysplasia (mFMD) have not been previously defined. Approach and Results: We studied 4 independent probands with the COL5A1 pathogenic variant c.1540G>A, p.(Gly514Ser) who presented with arterial aneurysms, dissections, tortuosity, and mFMD affecting multiple arteries. Arterial medial fibroplasia and smooth muscle cell disorganization were confirmed histologically. The COL5A1 c.1540G>A variant is predicted to be pathogenic in silico and absent in gnomAD. The c.1540G>A variant is on a shared 160.1 kb haplotype with 0.4% frequency in Europeans. Furthermore, exome sequencing data from a cohort of 264 individuals with mFMD were examined for COL5A1 variants. In this mFMD cohort, COL5A1 c.1540G>A and 6 additional relatively rare COL5A1 variants predicted to be deleterious in silico were identified and were associated with arterial dissections (P=0.005). CONCLUSIONS: COL5A1 c.1540G>A is the first recurring variant recognized to be associated with arterial dissections and mFMD. This variant presents with a phenotype reminiscent of vascular Ehlers-Danlos syndrome. A shared haplotype among probands supports the existence of a common founder. Relatively rare COL5A1 genetic variants predicted to be deleterious by in silico analysis were identified in ≈2.7% of mFMD cases, and as they were enriched in patients with arterial dissections, may act as disease modifiers. Molecular testing for COL5A1 should be considered in patients with a phenotype overlapping with vascular Ehlers-Danlos syndrome and mFMD.