Homotrimeric Type Research Articles

Establishment of homotrimer collagen type I signature and its association with clinical manifestation and tertiary lymphoid structures formation in liver cancer

Backgroundcollagen type I is a fundamental composition of extracellular matrix. Typically it exists in the form of a heterotrimer, consisting of two α1 chains encoded by COL1A1 and one α2 chain encoded by COL1A2. However, in cancer a homotrimeric form of collagen type I comprises three α1 chains encoded by COL1A1 was founded. There is still a lack of transcriptional and histologic methods for detecting homotrimeric collagen type I. Furthermore, a comprehensive analysis of the pan-cancer distribution pattern and clinical relevance of homotrimeric collagen type I is conspicuously absent. MethodUsing transcriptional and immunoflourance method, we established homocol signature, which is able to transcriptionally and histologically detect homotrimeric collagen type I. We investigated the diagnostic and prognostic potential of homocol as a novel cancer biomarker in a pan-cancer cohort. Furthermore, we assessed its association with clinical manifestations in a liver cancer cohort undergoing treatment at our institute. ResultHomotrimer Collagen Type I is predominantly expressed by cancer cells and is linked to several critical cancer hallmarks, particularly inflammatory response and proliferation. Survival analyses have indicated that a high Homocol expression is correlated with poor outcomes in most types of cancer studied. In terms of cancer detection, Homocol demonstrated strong performance in Receiver Operating Characteristic (ROC) analysis, with an Area Under Curve (AUC) of 0.83 for pan-cancer detection and between 0.72 and 0.99 for individual cancers.In cohorts undergoing PD1 treatment, we noted a higher presence of Homocol in the response group. In a Hepatocellular Carcinoma (HCC) clinical set, high Homocol expression was associated with an increased formation of intra-tumor tertiary lymphoid structures (TLS), larger tumor sizes, more advanced Barcelona Clinic Liver Cancer (BCLC) stages, higher microvascular invasion (MVI) grades, absence of a capsule, and an enriched para-tumor collagen presence. Conclusionour research has led to the development of a novel gene signature that facilitates the detection of Homotrimer Collagen Type I. This may greatly assist efforts in cancer detection, prognosis, treatment response prediction, and further research into Homotrimer Collagen Type I.

Collagen (I) homotrimer potentiates the osteogenesis imperfecta (oim) mutant allele and reduces survival in male mice.

ABSTRACTThe osteogenesis imperfecta murine (oim) model with solely homotrimeric (α1)3 type I collagen, owing to a dysfunctional α2(I) collagen chain, has a brittle bone phenotype, implying that the (α1)2(α2)1 heterotrimer is required for physiological bone function. Here, we comprehensively show, for the first time, that mice lacking the α2(I) chain do not have impaired bone biomechanical or structural properties, unlike oim homozygous mice. However, Mendelian inheritance was affected in male mice of both lines, and male mice null for the α2(I) chain exhibited age-related loss of condition. Compound heterozygotes were generated to test whether gene dosage was responsible for the less-severe phenotype of oim heterozygotes, after allelic discrimination showed that the oim mutant allele was not downregulated in heterozygotes. Compound heterozygotes had impaired bone structural properties compared to those of oim heterozygotes, albeit to a lesser extent than those of oim homozygotes. Hence, the presence of heterotrimeric type I collagen in oim heterozygotes alleviates the effect of the oim mutant allele, but a genetic interaction between homotrimeric type I collagen and the oim mutant allele leads to bone fragility.

Probing the gating mechanism of the mechanosensitive channel Piezo1 with the small molecule Yoda1

Piezo proteins are transmembrane ion channels which transduce many forms of mechanical stimuli into electrochemical signals. Their pore, formed by the assembly of three identical subunits, opens by an unknown mechanism. Here, to probe this mechanism, we investigate the interaction of Piezo1 with the small molecule agonist Yoda1. By engineering chimeras between mouse Piezo1 and its Yoda1-insensitive paralog Piezo2, we first identify a minimal protein region required for Yoda1 sensitivity. We next study the effect of Yoda1 on heterotrimeric Piezo1 channels harboring wild type subunits and Yoda1-insensitive mutant subunits. Using calcium imaging and patch-clamp electrophysiology, we show that hybrid channels harboring as few as one Yoda1-sensitive subunit exhibit Yoda1 sensitivity undistinguishable from homotrimeric wild type channels. Our results show that the Piezo1 pore remains fully open if only one subunit remains activated. This study sheds light on the gating and pharmacological mechanisms of a member of the Piezo channel family.

Soluble Activin Type IIB Receptor Decoy changes Gene Expression Profiles of Bone Cells in the OIM and not the G610C Mouse Model of Osteogenesis Imperfecta

Bone is a highly dynamic tissue which is continually remodeled by osteoblasts and osteoclasts, with regulation by osteocytes. In Osteogenesis Imperfecta (OI), a clinically heterogeneous disorder characterized by bone fragility, the functions of osteoblasts and osteoclasts are thought to be impaired. As a mechanosensitive tissue, bone responds to mechanical loads, some of the strongest of which are from skeletal muscles, by changing its shape.The inhibition of myostatin, a negative regulator of muscle growth and a member of the TGF‐β superfamily, has been shown to induce muscle growth via hypertrophy. In this study, we investigated the effect of pharmacologically inhibiting myostatin through treatment with the soluble activin receptor type IIB decoy molecule (sActRIIB‐mFc), focusing on the gene expression patterns of bone cells derived from two molecularly distinct mouse models of OI (osteogenesis imperfecta murine [OIM] vs. G610C). Because the molecular defects that give rise to the G610C and OIM models differ and muscle function is differentially impacted, we hypothesize that the two models may respond differently to sActRIIB‐mFc treatment. Homozygous mice (oim/oim) in the OIM model have a single nucleotide deletion in the COL1A2 gene that leads to functional null proα2 (I) collagen chains and the generation of homotrimeric type I collagen, whereas the G610C mouse, heterozygous for a missense mutation in the COL1A2 gene, has a glycine to cysteine substitution at the 610 position of the triple helical domain.At two months of age, WT, +/G610C, and oim/oim male mice were treated bi‐weekly either with vehicle (Tris‐buffered saline) or sActRIIB‐mFc for 8 weeks. Quantitative RT‐PCR analyses demonstrated that tibias from sActRIIB‐mFc treated oim/oim mice exhibited increased gene expression levels of the markers of osteoblast, osteoclast, and osteocyte function and differentiation analyzed. This contrasts with the significant changes seen in the femoral cortical bone microarchitecture and biomechanical integrity of sActRIIB‐mFc‐treated WT and +/G610C mice, which was not observed in the oim/oim mice.In conclusion, our findings indicate that sActRIIB‐mFc treatment has differential effects on the G610C and OIM mice, which may in part reflect differences in severity (severe in oim/oim vs. moderately severe in +/G610C), and/or their unique molecular defects.Understanding the molecular impact of therapeutics is critical in order to develop more effective and targeted treatment for OI.Support or Funding InformationLeda J. Sears TrustKansas City Area life Sciences Institute Patton Trust Research, Inc. University of Missouri Research Board GrantMarch of DimesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transforming growth factor-β1/Smad3-independent epithelial-mesenchymal transition in type I collagen glomerulopathy.

The glomerulofibrotic Col1a2-deficient mouse model demonstrates glomerular homotrimeric type I collagen deposition in mesangial and subendothelial spaces. In this report, we investigate the role of transforming growth factor β1 (TGF-β1) in myofibroblast activation and epithelial–mesenchymal transition (EMT) in this glomerulopathy. Immunohistochemical analyses of glomerular α-sma, desmin, vimentin, and proliferating cell nuclear antigen demonstrated parietal epithelial cell proliferation and EMT in late stages of the glomerulopathy in the Col1a2-deficient mice. Glomerular TGF-β1 RNA and protein were not elevated in 1- and 3-month-old mice as determined by quantitative reverse transcriptase-polymerase chain reaction and protein immunoassay analyses. To investigate further whether TGF-β1 plays a role in the glomerulopathy outside of the 1- and 3-month time periods, the Col1a2-deficient mice were bred with Smad3 knockout mice. If the glomerular fibrosis in the Col1a2-deficient mice is mediated by the TGF-β1/Smad3 transcription pathway, it was hypothesized that the resultant Col1a2-deficient/Smad3-deficient mice would exhibit attenuated glomerular homotrimer deposition. However, the Col1a2-deficient/Smad3-deficient kidneys were similarly affected as compared to age-matched Col1a2-deficient kidneys, suggesting that homotrimeric type I collagen deposition in the Col1a2-deficient mouse is independent of TGF-β1/Smad3 signaling. Deposition of homotrimeric type I collagen appears to be the initiating event in this glomerulopathy, providing evidence that EMT and myofibroblast activation occur following initiation, consistent with a secondary wound-healing response independent of TGF-β1.

Measurement of the serum B-cell-activating factor of the tumor necrosis factor family (BAFF) in patients with idiopathic thrombocytopenic purpura and its correlation with immunosuppressive therapy

Background The B-cell-activating factor of the tumor necrosis factor family (BAFF) is a homotrimeric type 2 transmembrane protein that also exists in a soluble form. It belongs to the family of tumor necrosis factor ligands and is expressed at the surfaces of myeloid cells and antigen-presenting cells and induces B-lymphocyte proliferation and immunoglobulin secretion. Aim of the study The aim of this study was to assess the serum BAFF level in patients with idiopathic thrombocytopenic purpura (ITP) and to determine the correlation of its level with response to immunosuppressive therapy (corticosteroids). Participants and methods The study included 60 participants: 40 patients with newly diagnosed ITP who were followed up for 3 months after immunosuppressive therapy (steroids), divided into responders and nonresponders, and 20 healthy control individuals. Results The serum BAFF level was lower in the controls (mean±SD 3.4±2.2 ng/ml) than the ITP patients (responders and nonresponders) before treatment (mean±SD 19.8±2.9 and 20.5±8.8 ng/ml, respectively) with a statistically highly significant difference ( P P Conclusion The serum BAFF level increases in patients with ITP and reverts to normal after treatment in responders but remains high in nonresponders. We recommend further randomized-controlled clinical trials exploring the role of drugs acting on BAFF in the treatment of autoimmune disorders.

Deficient degradation of homotrimeric type I collagen, α1(I)3 glomerulopathy in oim mice

Col1a2-deficient (oim) mice synthesize homotrimeric type I collagen due to nonfunctional proα2(I) collagen chains. Our previous studies revealed a postnatal, progressive type I collagen glomerulopathy in this mouse model, but the mechanism of the sclerotic collagen accumulation within the renal mesangium remains unclear. The recent demonstration of the resistance of homotrimeric type I collagen to cleavage by matrix metalloproteinases (MMPs), led us to investigate the role of MMP-resistance in the glomerulosclerosis of Col1a2-deficient mice. We measured the pre- and post-translational expression of type I collagen and MMPs in glomeruli from heterozygous and homozygous animals. Both the heterotrimeric and homotrimeric isotypes of type I collagen were equally present in whole kidneys of heterozygous mice by immunohistochemistry and biochemical analysis, but the sclerotic glomerular collagen was at least 95–98% homotrimeric, suggesting homotrimeric type I collagen is the pathogenic isotype of type I collagen in glomerular disease. Although steady-state MMP and Col1a1 mRNA levels increased with the disease progression, we found these changes to be a secondary response to the deficient clearance of MMP-resistant homotrimers. Increased renal MMP expression was not sufficient to prevent homotrimeric type I collagen accumulation.

ENU-induced missense mutation in the C-propeptide coding region of Col2a1 creates a mouse model of platyspondylic lethal skeletal dysplasia, Torrance type

The COL2A1 gene encodes the α1(II) chain of the homotrimeric type II collagen, the most abundant protein in cartilage. In humans, COL2A1 mutations create many clinical phenotypes collectively termed type II collagenopathies; however, the genetic basis of the phenotypic diversity is not well elucidated. Therefore, animal models corresponding to multiple type II collagenopathies are required. In this study we identified a novel Col2a1 missense mutation--c.44406A>C (p.D1469A)--produced by large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis in a mouse line. This mutation was located in the C-propeptide coding region of Col2a1 and in the positions corresponding to a human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). The phenotype was inherited as a semidominant trait. The heterozygotes were mildly but significantly smaller than wild-type mice. The homozygotes exhibited lethal skeletal dysplasias, including extremely short limbs, severe spondylar dysplasia, severe pelvic hypoplasia, and brachydactyly. As expected, these skeletal defects in the homozygotes were similar to those in PLSD-T patients. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by abnormally expanded rough endoplasmic reticulum (ER) and upregulation of ER stress-related genes, such as Grp94 and Chop, in chondrocytes. These findings suggested that the accumulation of mutant type II collagen in the ER and subsequent induction of ER stress are involved, at least in part in the PLSD-T-like phenotypes of the mutants. This mutant should serve as a good model for studying PLSD-T pathogenesis and the mechanisms that create the great diversity of type II collagenopathies.

Dietary Fluoride Restriction Does Not Alter Femoral Biomechanical Strength in col1a2-Deficient (oim) Mice with Type I Collagen Glomerulopathy ,

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease due primarily to mutations in the type I procollagen genes, COL1A1 and COL1A2, causing bone deformity and numerous lifetime fractures. OI murine (oim) model mice carry a mutation in the col1a2 gene causing aberrant production of homotrimeric type I collagen [α1(I)3], leading to bone fragility and glomerular accumulation of type I collagen. Previous studies demonstrated that heterozygous (+/oim) and homozygous (oim/oim) mice have elevated tibiae fluoride concentrations but reduced femoral biomechanics. However, it is unclear whether these 2 variables are causally related, because impaired renal function could reduce urinary fluoride excretion, thus elevating bone fluoride concentrations regardless of disease status. Our goal in this study was to determine whether dietary fluoride restriction would improve femoral biomechanics in oim mice. Wild-type, +/oim, and oim/oim mice were fed a control (5 mg/kg fluoride) or fluoride-restricted diet (0 mg/kg fluoride) for ~13 wk, at which time plasma and femora were analyzed for fluoride concentrations and bone biomechanical properties. In wild-type, +/oim, and oim/oim mice, dietary fluoride restriction reduced femoral fluoride burden by 54–74%, respectively (P < 0.05), without affecting glomerular collagen deposition. Oim/oim mice fed the fluoride-restricted diet had reduced material tensile strength (P < 0.05) compared with oim/oim mice fed the control diet. However, dietary fluoride restriction did not affect stiffness or whole bone femoral breaking strength, regardless of genotype. These data suggest that oim mice have reduced bone strength due to homotrimeric type I collagen, independent of bone fluoride content.

Carcinomas contain a matrix metalloproteinase-resistant isoform of type I collagen exerting selective support to invasion.

Collagen fibers affect metastasis in two opposing ways, by supporting invasive cells but also by generating a barrier to invasion. We hypothesized that these functions might be performed by different isoforms of type I collagen. Carcinomas are reported to contain alpha1(I)(3) homotrimers, a type I collagen isoform normally not present in healthy tissues, but the role of the homotrimers in cancer pathophysiology is unclear. In this study, we found that these homotrimers were resistant to all collagenolytic matrix metalloproteinases (MMP). MMPs are massively produced and used by cancer cells and cancer-associated fibroblasts for degrading stromal collagen at the leading edge of tumor invasion. The MMP-resistant homotrimers were produced by all invasive cancer cell lines tested, both in culture and in tumor xenografts, but they were not produced by cancer-associated fibroblasts, thereby comprising a specialized fraction of tumor collagen. We observed the homotrimer fibers to be resistant to pericellular degradation, even upon stimulation of the cells with proinflammatory cytokines. Furthermore, we confirmed an enhanced proliferation and migration of invasive cancer cells on the surface of homotrimeric versus normal (heterotrimeric) type I collagen fibers. In summary, our findings suggest that invasive cancer cells may use homotrimers for building MMP-resistant invasion paths, supporting local proliferation and directed migration of the cells whereas surrounding normal stromal collagens are cleaved. Because the homotrimers are universally secreted by cancer cells and deposited as insoluble, MMP-resistant fibers, they offer an appealing target for cancer diagnostics and therapy.

Mechanical properties of physiological and pathological models of collagen peptides investigated via steered molecular dynamics simulations

In this work we used molecular simulations to investigate the elastic properties of collagen single chain and triple helix with the aim of understanding its features starting from first principles. We analysed ideal collagen peptides, homotrimeric and heterotrimeric collagen type I and pathological models of collagen. Triple helices were found much more rigid than single chains, thus enlightening the important role of interchain stabilizing forces, like hydrophobic interaction and hydrogen bonds. We obtained Young's moduli close to 4.5 GPa for the ideal model of collagen and for the physiological heterotrimer, while the physiological homotrimer presented a Young's modulus of 2.51 GPa, that can be related to a mild form of Osteogenesis Imperfecta in which only the homotrimeric form of collagen type I is produced. Otherwise, the pathological model (presenting a glycine to alanine substitution) showed an elastic modulus of 4.32 GPa, thus only slightly lower than the ideal model. This suggests that this mutation only slightly affects the mechanical properties of the collagen molecule, but possibly acts on an higher scale, such as the packing of collagen fibrils.

Transplantation of human fetal mesenchymal stem cells improves glomerulopathy in a collagen type Iα2‐deficient mouse

Fetal mesenchymal stem cell (fetal MSC) therapy has potential to treat genetic diseases with early onset, including those affecting the kidney and urinary tract. A collagen type I alpha 2-deficient mouse has a deletion in the alpha2 chain of the procollagen type I gene, resulting in the synthesis of abnormal alpha1(I)(3) homotrimers, which replace normal alpha 1(I)2 alpha 2(I)1 heterotrimers and a glomerulopathy. We first confirmed that col1 alpha 2-deficient homozygous mice show abnormal collagen deposition in the glomeruli, which increases in frequency and severity with postnatal age. Intrauterine transplantation of human MSCs from first trimester fetal blood led postnatally to a reduction of abnormal homotrimeric collagen type I deposition in the glomeruli of 4-12 week-old col1 alpha 2-deficient mice. Using bioluminescence imaging, in situ hybridization and immunohistochemistry in transplanted col1 alpha 2-deficient mice, we showed that the damaged kidneys preferentially recruited donor cells in glomeruli, around mesangial cells. Real-time RT-PCR demonstrated that this effect was seen at an engraftment level of 1% of total cells in the kidney, albeit higher in glomeruli. We conclude that intrauterine transplantation of human fetal MSCs improves renal glomerulopathy in a collagen type I-deficient mouse model. These data support the feasibility of prenatal treatment for hereditary renal diseases.

Single‐nucleotide polymorphisms in the COL1A1 regulatory regions are associated with otosclerosis

Otosclerosis (MIM 166800) has a prevalence of 0.2-1% among white adults, making it the single most common cause of hearing impairment in this ethnic group. Although measles virus, hormones, human leukocyte antigen alleles and genetic factors have been implicated in the development of otosclerosis, its etiology remains unknown. In a focused effort to identify genetic factors in otosclerosis, we have mapped four disease loci (MIM 166800/605727/608244/608787); however, cloning the disease-causing genes in these intervals has not been successful. Here, we used a case-control study design to investigate the association between collagen type I genes and otosclerosis. We identified susceptibility and protective haplotypes in COL1A1 that are significantly associated with otosclerosis in the Caucasian population. These haplotypes alter reporter gene activity in an osteoblast cell line by affecting binding of transcription factors to cis-acting elements. Our data suggest that increased amounts of collagen alpha1(I) homotrimers are causally related to the development of otosclerosis. Consistent with this hypothesis, mouse mutants homozygous for a Col1a2 frameshift mutation on a C57BL/6J background that deposit only homotrimeric type I collagen have hearing loss.

Type I collagen glomerulopathy: postnatal collagen deposition follows glomerular maturation

In chronic renal disease, the progressive accumulation of collagen and other extracellular matrix proteins in the mesangium results in fibrosis, glomerulosclerosis, and eventual renal failure. Mice deficient in proalpha2(I) collagen are not only a model of osteogenesis imperfecta but also accumulate fibrillar homotrimeric type I collagen in the mesangium. This accumulation spreads to the subendothelial space in the peripheral capillary loops. Picosirius red staining of kidney sections demonstrates that in comparison to wild-type mice, Col1a2-deficient homozygous and heterozygous mice exhibit abnormal glomerular collagen deposition in a gene dosage-dependent manner. The glomerulopathy initiates during the first postnatal week, appears progressive following the pattern of glomerular maturation and results in albuminuria in severely affected animals. In situ hybridization revealed no gross differences in steady-state proalpha1(I) and proalpha2(I) collagen mRNA levels among the three genotypes. Quantitative reverse transcriptase-polymerase chain reaction, however, using whole kidney sections showed a twofold increase in steady-state proalpha1(I) collagen mRNA in 1-month homozygous Col1a2-deficient animals compared with wild-type and heterozygous animals. We suggest that glomerular collagen deposition seen in the osteogenesis imperfecta model mice is, in part, owing to pretranslational mechanisms and may represent an over compensation of wound healing.

Regulatory Role of FGF-2 on Type I Collagen Expression during Endothelial Mesenchymal Transformation

To investigate the regulatory role of FGF-2 on type I collagen expression during endothelial mesenchymal transformation (EMT). Corneal endothelial cells (CECs) treated with FGF-2 from the primary culture to the third passage were transformed and designated as fibroblastic CECs (fCECs). Steady state levels of both type I collagen RNAs were measured using reverse transcription-real-time PCR, and their half lives were determined in the presence of inhibitor of RNA synthesis. Limited proteolysis with pepsin was used to determine secretion of type I collagen. Protein-protein interaction was determined by coimmunoprecipitation, and subcellular localization was studied by immunofluorescence. fCECs were characterized by greatly stimulated proliferative potential, loss of contact inhibition, and multilayer fibroblastic cells. The steady state level of alpha1(I) collagen RNA was greatly upregulated through stabilization of the message in fCECs, whereas steady state level and half-life of the alpha2(I) collagen RNA were slightly increased compared with the corresponding levels in normal CECs. Of interest, fCECs predominantly secreted homotrimeric type I collagen, [alpha1(I)](3), with heterotrimeric type I collagen as a minor species. Type I collagen in fCECs was preferentially associated and colocalized with Hsp47 at Golgi apparatus as opposed to its association with protein disulfide isomerase in CECs. LY294002 (a specific PI 3-kinase inhibitor) greatly reduced the steady state levels and stability of alpha1(I) and alpha2(I) collagen RNAs and the secretion of type I collagen. FGF-2 directly mediates corneal EMT through the action of PI 3-kinase, which acts on posttranscriptional regulation by affecting the stability of type I collagen RNA.

Assembly of homotrimeric type XXI minicollagen by coexpression of prolyl 4-hydroxylase in stably transfected Drosophila melanogaster S2 cells

We established stably transfected insect cell lines containing cDNAs encoding the α and β subunits of human prolyl 4-hydroxylase in both Trichoplusia ni and Drosophila melanogaster S2 cells. The expression level and enzymatic activity of recombinant prolyl 4-hydroxylase produced in the Drosophila expression system were significantly higher than those produced in the T. ni system. We further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three α chains to form trimeric type XXI minicollagen, which comprises the intact C-terminal non-collagenous (NC1) and collagenous domain (COL1), in the Drosophila system. When minicollagen XXI was stably expressed in Drosophila S2 cells alone, negligible amounts of interchain disulfide-bonded trimers were detected in the culture media. However, minicollagen XXI was secreted as disulfide-bonded homotrimers by coexpression with prolyl 4-hydroxylase in the stably transfected Drosophila S2 cells. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient amounts of hydroxyproline to form thermal stable pepsin-resistant triple helices consisting of both interchain and non-interchain disulfide-bonded trimers. These results demonstrate that a sufficient amount of active prolyl 4-hydroxylase is required for the assembly of type XXI collagen triple helices in Drosophila cells and the trimeric assembly is governed by the C-terminal collagenous domain.

Alpha 2(I) collagen deficient oim mice have altered biomechanical integrity, collagen content, and collagen crosslinking of their thoracic aorta

Collagen and elastin are the primary determinants of vascular integrity, with elastin hypothesized to be the major contributor to aortic compliance and type I collagen the major contributor to aortic strength and stiffness. Type I collagen is normally heterotrimeric composed of two alpha1(I) and one alpha2(I) collagen chains, alpha1(I)(2)alpha2(I). Recent investigations have reported that patients with recessively inherited forms of Ehlers Danlos syndrome that fail to synthesize proalpha2(I) chains have increased risks of cardiovascular complications. To assess the role of alpha2(I) collagen in aortic integrity, we used the osteogenesis imperfecta model (oim) mouse. Oim mice, homozygous for a COL1A2 mutation, synthesize only homotrimeric type I collagen, alpha1(I)3. We evaluated thoracic aortas from 3-month-old oim, heterozygote, and wildtype mice biomechanically for circumferential breaking strength (Fmax) and stiffness (IEM), histologically for morphological differences, and biochemically for collagen content and crosslinking. Circumferential biomechanics of oim and heterozygote descending thoracic aortas demonstrated the anticipated reduced Fmax and IEM relative to wildtype mice. Histological analyses of oim descending aortas demonstrated reduced collagen staining relative to wildtype aortas suggesting decreased collagen content, which hydroxyproline analyses of ascending and descending oim aortas confirmed. These findings suggest the reduced oim thoracic aortic integrity correlates with the absence of the alpha2(I)collagen chains and in part with reduced collagen content. However, oim ascending aortas also demonstrated a significant increase in pyridinoline crosslinks/collagen molecule as compared to wildtype ascending aortas. The role of increased collagen crosslinks is uncertain; increased crosslinking may represent a compensatory mechanism for the decreased integrity.

Recognition of a single transmembrane degron by sequential quality control checkpoints.

To understand the relationship between conformational maturation and quality control-mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the alpha-subunit of the T-cell antigen receptor (TCRalpha) into the alpha-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only approximately 50% of nascent mutant HA chains (HA(++)) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining approximately 50% of nascent HA(++) chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA(++) molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HA(wt). These apparently native HA(++) molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane.

Novel collagen glomerulopathy in a homotrimeric type I collagen mouse (oim)

Oim/oim mice [osteogenesis imperfecta model; homozygous null for the proalpha2(I) collagen gene] synthesize exclusively the homotrimeric type I collagen isotype, alpha1(I)3, and are unable to synthesize the normal heterotrimeric type I collagen isotype, alpha1(I)2alpha2(I). Previous studies of the oim/oim mouse have focused on the musculoskeletal system, with no systematic evaluation of other organ systems. Multiple tissues from oim/oim, oim/+ (heterozygous) and +/+ (wild-type) mice were examined for gross and histological abnormalities. Tissues were stained with (1) hematoxylin and eosin (to assess lesion formation), (2) picrosirius red (collagen content), and (3) periodic acid methenamine silver (basement membrane). Kidneys were further evaluated ultrastructurally by electron microscopy and immunohistochemically with anti-alpha1(I) and anti-alpha1(III) collagen antibodies. Histological analyses revealed accumulations of picrosirius red-positive material, consistent with collagen, in glomeruli of 28/29 oim/oim mice, with no evidence of mesangial cell proliferation. Only the most severely affected animals had evidence of increased capillary basement membrane thickening or mild inflammation around the affected glomeruli. Electron microscopy confirmed the presence of fibrillar collagen. Immunohistochemistry with anti-alpha1(I) collagen antibodies confirmed accumulation of type I collagen in the oim/oim glomeruli. The +/+ and oim/+ kidneys had normal mesangium with no evidence of infiltration of collagenous material. This study demonstrates the first evidence, to our knowledge, of abnormal glomerular collagen deposition associated with a type I collagen defect. Further in vivo and in vitro studies are necessary to elucidate the mechanistic, functional, and pathological significance of the oim/oim collagen glomerulopathy.

The role of type I collagen in aortic wall strength with a homotrimeric [α1(I)] 3 collagen mouse model

Purpose: Elastin and collagen (types I and III) are the primary load-bearing elements in aortic tissue. Deficiencies and derangements in elastin and type III collagen have been associated with the development of aneurysmal disease. However, the role of type I collagen is less well defined. The purpose of this study was to define the role of type I collagen in maintaining biomechanical integrity in the thoracic aorta, with a mouse model that produces homotrimeric type I collagen [α1(I)] 3, rather than the normally present heterotrimeric [α1(I)] 2 α2(I) type I collagen isotype. Methods: Ascending and descending thoracic aortas from homozygous ( oim/oim ), heterozygous ( oim /+), and wildtype (+/+) mice were harvested. Circumferential and longitudinal load-extension curves were used as a means of determining maximum breaking strength (F max) and incremental elastic modulus (IEM). Histologic analyses and hydroxyproline assays were performed as a means of determining collagen organization and content. Results: Circumferentially, the ascending and descending aortas of oim / oim mice demonstrated significantly reduced F max, with an F max of only 60% and 23%, respectively, of wildtype mice aortas. Oim/oim descending aortas demonstrated significantly greater compliance (decreased IEM), and the ascending aortas also exhibited a trend toward increased compliance. Reduced breaking strength was also demonstrated with longitudinal extension of the descending aorta. Conclusion: The presence of homotrimeric type I collagen isotype (absence of α2(I) collagen) significantly weakens the aorta. This study demonstrates the integral role of type I collagen in the biomechanical and functional properties of the aorta and may help to elucidate the role of collagen in the development of aneurysmal aortic disease or dissection. (J Vasc Surg 2001;33:1263-70.)