A novel human iPSC model of COL4A1/A2 small vessel disease unveils a key pathogenic role of matrix metalloproteinases.

Cerebral small vessel disease (SVD) affects the small vessels in the brain and is a leading cause of stroke and dementia. Emerging evidence supports a role of the extracellular matrix (ECM), at the interface between blood and brain, in the progression of SVD pathology, but this remains poorly characterized. To address ECM role in SVD, we developed a co-culture model of mural and endothelial cells using human induced pluripotent stem cells from patients with COL4A1/A2 SVD-related mutations. This model revealed that these mutations induce apoptosis, migration defects, ECM remodeling, and transcriptome changes in mural cells. Importantly, these mural cell defects exert a detrimental effect on endothelial cell tight junctions through paracrine actions. COL4A1/A2 models also express high levels of matrix metalloproteinases (MMPs), and inhibiting MMP activity partially rescues the ECM abnormalities and mural cell phenotypic changes. These data provide a basis for targeting MMP as a therapeutic opportunity in SVD.

Vascular Collagen Type-IV in Hypertension and Cerebral Small Vessel Disease.

BACKGROUND: Cerebral small vessel disease (SVD) is common in older people and causes lacunar stroke and vascular cognitive impairment. Risk factors include old age, hypertension and variants in the genes COL4A1/COL4A2 encoding collagen alpha-1(IV) and alpha-2(IV), here termed collagen-IV, which are core components of the basement membrane. We tested the hypothesis that increased vascular collagen-IV associates with clinical hypertension and with SVD in older persons and with chronic hypertension in young and aged primates and genetically hypertensive rats. METHODS: We quantified vascular collagen-IV immunolabeling in small arteries in a cohort of older persons with minimal Alzheimer pathology (N=52; 21F/31M, age 82.8±6.95 years). We also studied archive tissue from young (age range 6.2-8.3 years) and older (17.0-22.7 years) primates (M mulatta) and compared chronically hypertensive animals (18 months aortic stenosis) with normotensives. We also compared genetically hypertensive and normotensive rats (aged 10-12 months). RESULTS: Collagen-IV immunolabeling in cerebral small arteries of older persons was negatively associated with radiological SVD severity (ρ: -0.427, P=0.005) but was not related to history of hypertension. General linear models confirmed the negative association of lower collagen-IV with radiological SVD (P<0.017), including age as a covariate and either clinical hypertension (P<0.030) or neuropathological SVD diagnosis (P<0.022) as fixed factors. Reduced vascular collagen-IV was accompanied by accumulation of fibrillar collagens (types I and III) as indicated by immunogold electron microscopy. In young and aged primates, brain collagen-IV was elevated in older normotensive relative to young normotensive animals (P=0.029) but was not associated with hypertension. Genetically hypertensive rats did not differ from normotensive rats in terms of arterial collagen-IV. CONCLUSIONS: Our cross-species data provide novel insight into sporadic SVD pathogenesis, supporting insufficient (rather than excessive) arterial collagen-IV in SVD, accompanied by matrix remodeling with elevated fibrillar collagen deposition. They also indicate that hypertension, a major risk factor for SVD, does not act by causing accumulation of brain vascular collagen-IV.

Four decades in the making: Collagen III and mechanisms of vascular Ehlers Danlos Syndrome.

Vascular Ehlers Danlos (vEDS) syndrome is a severe multi-systemic connective tissue disorder characterized by risk of dissection and rupture of the arteries, gastro-intestinal tract and gravid uterus. vEDS is caused by mutations in COL3A1, that encodes the alpha 1 chain of type III collagen, which is a major extracellular matrix component of the vasculature and hollow organs. The first causal mutations were identified in the 1980s but progress in our understanding of the pathomolecular mechanisms has been limited. Recently, the application of more refined animal models combined with global omics approaches has yielded important new insights both in terms of disease mechanisms and potential for therapeutic intervention. However, it is also becoming apparent that vEDS is a complex disorder in terms of its molecular disease mechanisms with a poorly understood allelic and mechanistic heterogeneity. In this brief review we will focus our attention on the disease mechanisms of COL3A1 mutations and vEDS, and recent progress in therapeutic approaches using animal models.

The role of basement membranes in cardiac biology and disease.

Basement membranes (BMs) are highly specialised extracellular matrix (ECM) structures that within the heart underlie endothelial cells (ECs) and surround cardiomyocytes and vascular smooth muscle cells. They generate a dynamic and structurally supportive environment throughout cardiac development and maturation by providing physical anchorage to the underlying interstitium, structural support to the tissue, and by influencing cell behaviour and signalling. While this provides a strong link between BM dysfunction and cardiac disease, the role of the BM in cardiac biology remains under-researched and our understanding regarding the mechanistic interplay between BM defects and their morphological and functional consequences remain important knowledge-gaps. In this review, we bring together emerging understanding of BM defects within the heart including in common cardiovascular pathologies such as contractile dysfunction and highlight some key questions that are now ready to be addressed.

Identification of an Altered Matrix Signature in Kidney Aging and Disease.

BACKGROUND: Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking. METHODS: Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features. RESULTS: Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets. CONCLUSIONS: This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair.

Rare Missense Functional Variants at COL4A1 and COL4A2 in Sporadic Intracerebral Hemorrhage.

ObjectiveTo test the genetic contribution of rare missense variants in COL4A1 and COL4A2 in which common variants are genetically associated with sporadic intracerebral hemorrhage (ICH), we performed rare variant analysis in multiple sequencing data for the risk for sporadic ICH.MethodsWe performed sequencing across 559Kbp at 13q34 including COL4A1 and COL4A2 among 2,133 individuals (1,055 ICH cases; 1,078 controls) in US-based and 1,492 individuals (192 ICH cases; 1,189 controls) from Scotland-based cohorts, followed by sequence annotation, functional impact prediction, genetic association testing, and in silico thermodynamic modeling.ResultsWe identified 107 rare nonsynonymous variants in sporadic ICH, of which two missense variants, rs138269346 (COL4A1I110T) and rs201716258 (COL4A2H203L), were predicted to be highly functional and occurred in multiple ICH cases but not in controls from the US-based cohort. The minor allele of rs201716258 was also present in Scottish ICH patients, and rs138269346 was observed in two ICH-free controls with a history of hypertension and myocardial infarction. Rs138269346 was nominally associated with non-lobar ICH risk (P=0.05), but not with lobar ICH (P=0.08), while associations between rs201716258 and ICH subtypes were non-significant (P>0.12). Both variants were considered pathogenic based on minor allele frequency (<0.00035 in EUR), predicted functional impact (deleterious or probably damaging), and in silico modeling studies (substantially altered physical length and thermal stability of collagen).ConclusionsWe identified rare missense variants in COL4A1/A2 in association with sporadic ICH. Our annotation and simulation studies suggest that these variants are highly functional and may represent targets for translational follow-up.

Global proteomic analysis of extracellular matrix in mouse and human brain highlights relevance to cerebrovascular disease.

The extracellular matrix (ECM) is a key interface between the cerebrovasculature and adjacent brain tissues. Deregulation of the ECM contributes to a broad range of neurological disorders. However, despite this importance, our understanding of the ECM composition remains very limited mainly due to difficulties in its isolation. To address this, we developed an approach to extract the cerebrovascular ECM from mouse and human post-mortem normal brain tissues. We then used mass spectrometry with off-line high-pH reversed-phase fractionation to increase the protein detection. This identified more than 1000 proteins in the ECM-enriched fraction, with > 66% of the proteins being common between the species. We report 147 core ECM proteins of the human brain vascular matrisome, including collagens, laminins, fibronectin and nidogens. We next used network analysis to identify the connection between the brain ECM proteins and cerebrovascular diseases. We found that genes related to cerebrovascular diseases, such as COL4A1, COL4A2, VCAN and APOE were significantly enriched in the cerebrovascular ECM network. This provides unique mechanistic insight into cerebrovascular disease and potential drug targets. Overall, we provide a powerful resource to study the functions of brain ECM and highlight a specific role for brain vascular ECM in cerebral vascular disease.

Basement membrane stiffness determines metastases formation.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts.

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin ß1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells.

Basement membrane collagens and disease mechanisms.

Basement membranes (BMs) are specialised extracellular matrix (ECM) structures and collagens are a key component required for BM function. While collagen IV is the major BM collagen, collagens VI, VII, XV, XVII and XVIII are also present. Mutations in these collagens cause rare multi-systemic diseases but these collagens have also been associated with major common diseases including stroke. Developing treatments for these conditions will require a collective effort to increase our fundamental understanding of the biology of these collagens and the mechanisms by which mutations therein cause disease. Novel insights into pathomolecular disease mechanisms and cellular responses to these mutations has been exploited to develop proof-of-concept treatment strategies in animal models. Combined, these studies have also highlighted the complexity of the disease mechanisms and the need to obtain a more complete understanding of these mechanisms. The identification of pathomolecular mechanisms of collagen mutations shared between different disorders represent an attractive prospect for treatments that may be effective across phenotypically distinct disorders.

4-Sodium phenyl butyric acid has both efficacy and counter-indicative effects in the treatment of Col4a1 disease.

Mutations in the collagen genes COL4A1 and COL4A2 cause Mendelian eye, kidney and cerebrovascular disease including intracerebral haemorrhage (ICH), and common collagen IV variants are a risk factor for sporadic ICH. COL4A1 and COL4A2 mutations cause endoplasmic reticulum (ER) stress and basement membrane (BM) defects, and recent data suggest an association of ER stress with ICH due to a COL4A2 mutation. However, the potential of ER stress as a therapeutic target for the multi-systemic COL4A1 pathologies remains unclear. We performed a preventative oral treatment of Col4a1 mutant mice with the chemical chaperone phenyl butyric acid (PBA), which reduced adult ICH. Importantly, treatment of adult mice with the established disease also reduced ICH. However, PBA treatment did not alter eye and kidney defects, establishing tissue-specific outcomes of targeting Col4a1-derived ER stress, and therefore this treatment may not be applicable for patients with eye and renal disease. While PBA treatment reduced ER stress and increased collagen IV incorporation into BMs, the persistence of defects in BM structure and reduced ability of the BM to withstand mechanical stress indicate that PBA may be counter-indicative for pathologies caused by matrix defects. These data establish that treatment for COL4A1 disease requires a multipronged treatment approach that restores both ER homeostasis and matrix defects. Alleviating ER stress is a valid therapeutic target for preventing and treating established adult ICH, but collagen IV patients will require stratification based on their clinical presentation and mechanism of their mutations.

Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology.

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.

The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV α5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations.

INTRODUCTION: X-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations. METHODS: Dermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV α5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL2) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid. RESULTS: Both COL4A5 missense variants were associated with reduced proliferation rates on day 6 (P = 0.01 and P = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels (P < 0.01), and reduced ER size (P < 0.01 by EM and P < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P < 0.01). Extracellular collagen IV α5 chain was increased in the M1 line only (P = 0.06). DISCUSSION: Sodium 4-phenylbutyrate increases collagen IV α5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV α5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.

Disruption of a miR-29 binding site leading to COL4A1 upregulation causes pontine autosomal dominant microangiopathy with leukoencephalopathy.

OBJECTIVE: Cerebral small vessel disease (cSVD) is a heterogeneous group of disorders. Screening of known cSVD genes identifies the causative mutation in <15% of familial cSVD cases. We sought to identify novel causes of cSVD. METHODS: We used linkage analysis and exome sequencing to identify the causal mutation in a French cSVD family. The identified candidate gene was then screened in 202 cSVD unrelated probands, including 1 proband from the first reported pontine autosomal dominant microangiopathy with leukoencephalopathy (PADMAL) family. Sanger sequencing was used to confirm variants in all mutated probands and analyze their segregation in probands' relatives. Mutation consequences were assessed with luciferase reporter assays and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: A candidate heterozygous variant located in a predicted miR-29 microRNA binding site, within the 3' untranslated region of COL4A1, was identified in the large French cSVD family. Five additional unrelated probands, including the PADMAL proband, harbored heterozygous variants in this microRNA binding site. Variants cosegregated with the affected phenotype, and cumulative logarithm of odds score reached 6.03, establishing linkage to this locus. A highly significant difference was observed when comparing the number of variants within this binding site in cases and controls (p = 1.77 × 10E-12). RT-qPCR analyses of patients' primary fibroblasts and luciferase reporter assays strongly favor an upregulation of COL4A1 mediated by disruption of miR-29 binding to its target site. Magnetic resonance imaging features were characterized by the presence of multiple pontine infarcts in all symptomatic mutation carriers. INTERPRETATION: Mutations upregulating COL4A1 expression lead to PADMAL, a severe early onset ischemic cSVD, distinct from the various phenotypes associated with COL4A1 missense glycine mutations. Ann Neurol 2016;80:741-753.

ER stress and basement membrane defects combine to cause glomerular and tubular renal disease resulting from Col4a1 mutations in mice.

Collagen IV is a major component of basement membranes, and mutations in COL4A1, which encodes collagen IV alpha chain 1, cause a multisystemic disease encompassing cerebrovascular, eye and kidney defects. However, COL4A1 renal disease remains poorly characterized and its pathomolecular mechanisms are unknown. We show that Col4a1 mutations in mice cause hypotension and renal disease, including proteinuria and defects in Bowman's capsule and the glomerular basement membrane, indicating a role for Col4a1 in glomerular filtration. Impaired sodium reabsorption in the loop of Henle and distal nephron despite elevated aldosterone levels indicates that tubular defects contribute to the hypotension, highlighting a novel role for the basement membrane in vascular homeostasis by modulation of the tubular response to aldosterone. Col4a1 mutations also cause diabetes insipidus, whereby the tubular defects lead to polyuria associated with medullary atrophy and a subsequent reduction in the ability to upregulate aquaporin 2 and concentrate urine. Moreover, haematuria, haemorrhage and vascular basement membrane defects confirm an important vascular component. Interestingly, although structural and compositional basement membrane defects occurred in the glomerulus and Bowman's capsule, no tubular basement membrane defects were detected. By contrast, medullary atrophy was associated with chronic ER stress, providing evidence for cell-type-dependent molecular mechanisms of Col4a1 mutations. These data show that both basement membrane defects and ER stress contribute to Col4a1 renal disease, which has important implications for the development of treatment strategies for collagenopathies.

Common variation in COL4A1/COL4A2 is associated with sporadic cerebral small vessel disease.

OBJECTIVES: We hypothesized that common variants in the collagen genes COL4A1/COL4A2 are associated with sporadic forms of cerebral small vessel disease. METHODS: We conducted meta-analyses of existing genotype data among individuals of European ancestry to determine associations of 1,070 common single nucleotide polymorphisms (SNPs) in the COL4A1/COL4A2 genomic region with the following: intracerebral hemorrhage and its subtypes (deep, lobar) (1,545 cases, 1,485 controls); ischemic stroke and its subtypes (cardioembolic, large vessel disease, lacunar) (12,389 cases, 62,004 controls); and white matter hyperintensities (2,733 individuals with ischemic stroke and 9,361 from population-based cohorts with brain MRI data). We calculated a statistical significance threshold that accounted for multiple testing and linkage disequilibrium between SNPs (p < 0.000084). RESULTS: Three intronic SNPs in COL4A2 were significantly associated with deep intracerebral hemorrhage (lead SNP odds ratio [OR] 1.29, 95% confidence interval [CI] 1.14-1.46, p = 0.00003; r(2) > 0.9 between SNPs). Although SNPs associated with deep intracerebral hemorrhage did not reach our significance threshold for association with lacunar ischemic stroke (lead SNP OR 1.10, 95% CI 1.03-1.18, p = 0.0073), and with white matter hyperintensity volume in symptomatic ischemic stroke patients (lead SNP OR 1.07, 95% CI 1.01-1.13, p = 0.016), the direction of association was the same. There was no convincing evidence of association with white matter hyperintensities in population-based studies or with non-small vessel disease cerebrovascular phenotypes. CONCLUSIONS: Our results indicate an association between common variation in the COL4A2 gene and symptomatic small vessel disease, particularly deep intracerebral hemorrhage. These findings merit replication studies, including in ethnic groups of non-European ancestry.

Proteome-based systems biology analysis of the diabetic mouse aorta reveals major changes in fatty acid biosynthesis as potential hallmark in diabetes mellitus-associated vascular disease.

BACKGROUND: Macrovascular complications of diabetes mellitus are a major risk factor for cardiovascular morbidity and mortality. Currently, studies only partially described the molecular pathophysiology of diabetes mellitus-associated effects on vasculature. However, better understanding of systemic effects is essential in unraveling key molecular events in the vascular tissue responsible for disease onset and progression. METHODS AND RESULTS: Our overall aim was to get an all-encompassing view of diabetes mellitus-induced key molecular changes in the vasculature. An integrative proteomic and bioinformatics analysis of data from aortic vessels in the low-dose streptozotocin-induced diabetic mouse model (10 animals) was performed. We observed pronounced dysregulation of molecules involved in myogenesis, vascularization, hypertension, hypertrophy (associated with thickening of the aortic wall), and a substantial reduction of fatty acid storage. A novel finding is the pronounced downregulation of glycogen synthase kinase-3ß (Gsk3ß) and upregulation of molecules linked to the tricarboxylic acid cycle (eg, aspartate aminotransferase [Got2] and hydroxyacid-oxoacid transhydrogenase [Adhfe1]). In addition, pathways involving primary alcohols and amino acid breakdown are altered, potentially leading to ketone-body production. A number of these findings were validated immunohistochemically. Collectively, the data support the hypothesis that in this diabetic model, there is an overproduction of ketone-bodies within the vessels using an alternative tricarboxylic acid cycle-associated pathway, ultimately leading to the development of atherosclerosis. CONCLUSIONS: Streptozotocin-induced diabetes mellitus in animals leads to a reduction of fatty acid biosynthesis and an upregulation of an alternative ketone-body formation pathway. This working hypothesis could form the basis for the development of novel therapeutic intervention and disease management approaches.

Chemical chaperone treatment reduces intracellular accumulation of mutant collagen IV and ameliorates the cellular phenotype of a COL4A2 mutation that causes haemorrhagic stroke.

Haemorrhagic stroke accounts for ∼20% of stroke cases and porencephaly is a clinical consequence of perinatal cerebral haemorrhaging. Here, we report the identification of a novel dominant G702D mutation in the collagen domain of COL4A2 (collagen IV alpha chain 2) in a family displaying porencephaly with reduced penetrance. COL4A2 is the obligatory protein partner of COL4A1 but in contrast to most COL4A1 mutations, the COL4A2 mutation does not lead to eye or kidney disease. Analysis of dermal biopsies from a patient and his unaffected father, who also carries the mutation, revealed that both display basement membrane (BM) defects. Intriguingly, defective collagen IV incorporation into the dermal BM was observed in the patient only and was associated with endoplasmic reticulum (ER) retention of COL4A2 in primary dermal fibroblasts. This intracellular accumulation led to ER stress, unfolded protein response activation, reduced cell proliferation and increased apoptosis. Interestingly, the absence of ER retention of COL4A2 and ER stress in cells from the unaffected father indicate that accumulation and/or clearance of mutant COL4A2 from the ER may be a critical modifier for disease development. Our analysis also revealed that mutant collagen IV is degraded via the proteasome. Importantly, treatment of patient cells with a chemical chaperone decreased intracellular COL4A2 levels, ER stress and apoptosis, demonstrating that reducing intracellular collagen accumulation can ameliorate the cellular phenotype of COL4A2 mutations. Importantly, these data highlight that manipulation of chaperone levels, intracellular collagen accumulation and ER stress are potential therapeutic options for collagen IV diseases including haemorrhagic stroke.

Tendon is covered by a basement membrane epithelium that is required for cell retention and the prevention of adhesion formation.

The ability of tendons to glide smoothly during muscle contraction is impaired after injury by fibrous adhesions that form between the damaged tendon surface and surrounding tissues. To understand how adhesions form we incubated excised tendons in fibrin gels (to mimic the homeostatic environment at the injury site) and assessed cell migration. We noticed cells exiting the tendon from only the cut ends. Furthermore, treatment of the tendon with trypsin resulted in cell extravagation from the shaft of the tendons. Electron microscopy and immunolocalisation studies showed that the tendons are covered by a novel cell layer in which a collagen type IV/laminin basement membrane (BM) overlies a keratinised epithelium. PCR and western blot analyses confirmed the expression of laminin ß1 in surface cells, only. To evaluate the cell retentive properties of the BM in vivo we examined the tendons of the Col4a1(+/Svc) mouse that is heterozygous for a G-to-A transition in the Col4a1 gene that produces a G1064D substitution in the α1(IV) chain of collagen IV. The flexor tendons had a discontinuous BM, developed fibrous adhesions with overlying tissues, and were acellular at sites of adhesion formation. In further experiments, tenotomy of wild-type mice resulted in expression of laminin throughout the adhesion. In conclusion, we show the existence of a novel tendon BM-epithelium that is required to prevent adhesion formation. The Col4a1(+/Svc) mouse is an effective animal model for studying adhesion formation because of the presence of a structurally-defective collagen type IV-containing BM.