Background: ␣1(V) is an extensively modified collagen chain important in disease. Results: Comprehensive mapping of ␣1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets. Conclusion:The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties. Significance: Positions, numbers, and occupancy of modified sites can provide insights into ␣1(V) biological properties.

Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2 À/À homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2 À/À embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2 À/À and Col5a1 À/À fibril formation and embryonic survival suggest that a1(V) 3 homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of a2(V) chains, serve functional roles that partially compensate for loss of the most common collagen V isoform. Col5a2 þ/À adults have skin with marked hyperextensibility and reduced tensile strength at high strain but not at low strain. Col5a2 þ/À adults also have aortas with increased compliance and reduced tensile strength. Results thus suggest that COL5A2 þ/À humans, although unlikely to present with frank classic Ehlers-Danlos syndrome, are likely to have fragile connective tissues with increased susceptibility to trauma and certain chronic pathologic conditions. Collagen V is a low-abundance fibrillar collagen widely distributed in vertebrate tissues as a1(V) 2 a2(V) heterotrimers, 1 which are incorporated into growing fibrils with the more abundant collagen I and involved in regulating the geometry and tensile strength of the resulting collagen I/V heterotypic fibrils. 2,3 Mutations in the genes encoding either the a1(V) 4 or a2(V) 5 chain can result in the human heritable connective tissue disorder classic Ehlers-Danlos syndrome (cEDS), clinical hallmarks of which include skin hyperextensibility, atrophic scarring, and joint hypermobility, with patients also often presenting with easy bruising and bleeding. 6 At the molecular level, the collagen fibrils of cEDS skin have variability in diameter not seen in normal skin and include large diameter collagen fibril aggregates with abnormal cauliflower-like shapes when viewed in cross section. 6 Deficits in the tensile strength of cEDS collagen fibrils are inferred from the hyperextensibility and fragility of cEDS skin and the hypermobility of cEDS joints.Most cEDS cases that have been characterized at the molecular level are heterozygous for null alleles of the a1(V) chain gene COL5A1, 7 resulting in the deposition of haploinsufficient levels of normal collagen V in tissues, with excess a2(V) chains unable to form stable triple helical molecules or be incorporated into the extracellular matrix (ECM). 8

Classic Ehlers-Danlos syndrome (cEDS) is characterized by fragile, hyperextensible skin and hypermobile joints. cEDS can be caused by heterozygosity for missense mutations in genes COL5A2 and COL5A1, which encode the α2(V) and α1(V) chains, respectively, of collagen V, and is most often caused by COL5A1 null alleles. However, COL5A2 null alleles have yet to be associated with cEDS or other human pathologies. We previously showed that mice homozygous null for the α2(V) gene Col5a2 are early embryonic lethal, whereas haploinsufficiency caused aberrancies of adult skin, but not a frank cEDS-like phenotype, as skin hyperextensibility at low strain and dermal cauliflower-contoured collagen fibril aggregates, two cEDS hallmarks, were absent. Herein, we show that ubiquitous postnatal Col5a2 knockdown results in pathognomonic dermal cauliflower-contoured collagen fibril aggregates, but absence of skin hyperextensibility, demonstrating these cEDS hallmarks to arise separately from loss of collagen V roles in control of collagen fibril growth and nucleation events, respectively. Col5a2 knockdown also led to loss of dermal white adipose tissue (WAT) and markedly decreased abdominal WAT that was characterized by miniadipocytes and increased collagen deposition, suggesting α2(V) to be important to WAT development/maintenance. More important, Col5a2 haploinsufficiency markedly increased the incidence and severity of abdominal aortic aneurysms, and caused aortic arch ruptures and dissections, indicating that α2(V) chain deficits may play roles in these pathologies in humans.

We have shown previously that collagen V (col(V)) autoimmunity is a consistent feature of atherosclerosis in human coronary artery disease and in the Apoe ؊/؊ mouse model. We have also shown sensitization of Apoe ؊/؊ mice with col(V) to markedly increase the atherosclerotic burden, providing evidence of a causative role for col(V) autoimmunity in atherosclerotic pathogenesis. Here we sought to determine whether induction of immune tolerance to col(V) might ameliorate atherosclerosis, providing further evidence for a causal role for col(V) autoimmunity in atherogenesis and providing insights into the potential for immunomodulatory therapeutic interventions. Mucosal inoculation successfully induced immune tolerance to col(V) with an accompanying reduction in plaque burden in Ldlr ؊/؊ mice on a high-cholesterol diet. The results therefore demonstrate that inoculation with col(V) can successfully ameliorate the atherosclerotic burden, suggesting novel approaches for therapeutic interventions. Surprisingly, tolerance and reduced atherosclerotic burden were both dependent on the recently described IL-35 and not on IL-10, the immunosuppressive cytokine usually studied in the context of induced tolerance and amelioration of atherosclerotic symptoms. In addition to the above, using recombinant protein fragments, we were able to localize two epitopes of the ␣1(V) chain involved in col(V) autoimmunity in atherosclerotic Ldlr ؊/؊ mice, suggesting future courses of experimentation for the characterization of such epitopes.Atherosclerosis underlies coronary artery disease (CAD) 3 and stroke, major global causes of death (1), and is a chronic inflammatory disease that is modulated by both innate and adaptive immune pathways. It is increasingly accepted that autoimmunity constitutes some portion of the pathological processes underlying atherosclerosis, with oxidized LDLs, native lipoproteins, and heat shock proteins identified as autoantigens involved in atherogenesis (1-8). Recognition of the autoimmune aspects of atherosclerosis has suggested the possibility of employing immunomodulatory approaches to ameliorate symptoms. That such an approach is feasible has been borne out in studies in which blockage of T cells reactive to native lipoprotein ApoB100 (4) or mucosal or subcutaneous immunization with HSP65 (9, 10), oxidized LDL (11), native ␤2-glycoprotein I (12), or apolipoprotein B-100 peptide (13, 14) have been shown to be atheroprotective. Collagens can compose up to 60% of the total protein content in atherosclerotic plaques (15) and stimulate the growth and inflammation of atheromas (16). We have shown previously that interleukin 17-dependent autoimmunity to type V collagen col(V)) is a consistent feature of atherosclerosis in advanced CAD in humans and in Apoe Ϫ/Ϫ mice on a high-fat diet (17). Moreover, the same study provided evidence of a causative role for col(V) autoimmunity in the pathogenesis of atherosclerosis because sensitization of Apoe Ϫ/Ϫ mice on normal chow to col(V) has been shown to markedly increase the ...

Th17-dependent autoimmune responses can develop after heart or lung transplantation, and are associated with fibro-obliterative forms of chronic rejection. However, the specific self-antigens involved are typically different from those associated with autoimmune disease. To investigate the basis of these responses, we questioned whether removal of Tregs or blockade of function reveals a similar auto-antigen bias. We found that Th17 cells specific for collagen type V (Col V), kα-1-tubulin, and vimentin were present in healthy, adult PBMC, cord blood, and fetal thymus. Using synthetic peptides and recombinant fragments of the Col V triple helical region (α1V), we compared Th17 cells from healthy donors with Th17 cells from Col V-reactive heart and lung patients. While the latter responded well to α1(V) fragments and peptides in a DR–restricted fashion, Th17 cells from healthy individuals responded in a DR-restricted fashion to fragments, but not to peptides. Col V, kα-1-tubulin, and vimentin are preferred targets of a highly conserved, hitherto unknown, pre-existing Th17 response that is MHCII-restricted. These data suggest that autoimmunity after heart and lung transplantation may result from dysregulation of an intrinsic mechanism controlling airway and vascular homeostasis.

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Background: The mTOR (mechanistic target of rapamycin) pathway is a complex signaling cascade that regulates cellular growth, proliferation, metabolism, and survival. Although activation of mTOR signaling has been linked to atherosclerosis, its direct role in lesion progression and in plaque macrophages remains poorly understood. We previously demonstrated that mTORC1 (mTOR complex 1) activation promotes atherogenesis through inhibition of autophagy and increased apoptosis in macrophages. Methods: Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. Results: In contrast to the atheroprotective effect seen with blockade of macrophage mTORC1, macrophage-specific mTORC2-deficient mice exhibit an atherogenic phenotype, with larger, more complex lesions and increased cell death. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 (forkhead box protein O1) transcription factor, leading to suppression of proinflammatory pathways, especially the inflammasome/IL (interleukin)-1β response, a key mediator of vascular inflammation and atherosclerosis. In addition, administration of FoxO1 inhibitors efficiently rescued the proinflammatory response caused by mTORC2 deficiency both in vitro and in vivo. Interestingly, collective deletion of macrophage mTOR, which ablates mTORC1- and mTORC2-dependent pathways, leads to minimal change in plaque size or complexity, reflecting the balanced yet opposing roles of these signaling arms. Conclusions: Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.

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