Collagen Α1 Chain Research Articles

Structure of a full-length cDNA clone for the pro-α1(V/XI) collagen chain of red seabream

The cDNA of type V/XI collagen α1 (rsCOL) chain has been isolated from cells established from eyed-period eggs of red seabream, Pagrus major, and sequenced. The amino acid sequence deduced from red seabream α1(V/XI) chain resembles that of type XI collagen α1 chain. On the other hand, tissue distribution of rsCOL resembles that of type V collagen based on RT–PCR analysis. This is the first report of the cloning of the full-length cDNA of type V/XI collagen α1 chain from fish.

COL9A2 Allelotypes in Intervertebral Disc Disease

An allelic variation of the COL9A2 gene encoding the α2-chain of collagen IX has recently been identified as a genetic risk factor for intervertebral disc prolapse, resulting in a tryptophane (Trp) substitution at position 326 of the protein. To enable quick screening of a large population we established a single enzyme (BsmFI) restriction assay which was validated by screening disc tissue samples of 250 patients (age, 47.1 ± 13.7 years). Positive results were confirmed by nucleotide sequencing. The Trp allele was found in three patients (1.2%) who suffered from their first prolapse and were significantly older (70.7 ± 8.5 years) than the other 247 patients. Since the substitution affects a domain covalently linked to collagen II fibrils, we conclude that this allelotype may contribute to reduced collagen crosslinking, disc instability and eventually prolapse in the elderly.

Development and evaluation of C-telopeptide enzyme-linked immunoassay for measurement of bone resorption in mouse serum

The mouse is increasingly being used as an animal model for the study of skeletal phenotypes in humans, mainly because of the ease of genetic manipulation. Biochemical markers of bone metabolism provide a valuable parameter for the assessment of skeletal metabolism. In the mouse model, assays for bone formation have been available for a long time; however, little is known about bone resorption markers. The present study describes the development of a serum C-telopeptide enzyme-linked immunoassay (ELISA), which measures degradation products of type I collagen that are generated by osteoclastic bone resorption. The C-telopeptide ELISA uses affinity-purified antibodies generated against human sequence DFSFLPQPPQEKAHDGGR. The epitope involves an amino acid sequence, which is identical in the mouse and human C-terminal peptide of type I collagen (α1 chain). Sensitivity of the ELISA used was <0.1 ng/mL. The average intra- (n = 10) and interassay (n = 8) coefficient of variation for two controls was <12%. The average dilution and spike recovery rates were 98% and 97%, respectively. Application of the ELISA to measure C-telopeptide in 3–4-week postovariectomized (ovx) C57BL/6J (B6) mice (n = 9 or 10) showed a 45% higher C-telopeptide concentration than the sham-operated mice. Treatment of ovx mice with estradiol (400 μg/kg body weight) or alendronate (1.0 mg/kg body weight) resulted in a 20%–50% decrease in C-telopeptide levels compared to the vehicle-treated ovx group. In addition, B6 mice fed a calcium-deficient diet (0.01% calcium) showed a 50% higher C-telopeptide concentration compared to the B6 mice receiving a normal diet (0.6% calcium). In conclusion, the C-telopeptide ELISA exhibited acceptable analytical performance and sufficient discriminatory power to show expected directional changes in the rate of bone resorption following ovariectomy, ovx plus estradiol or alendronate treatment, and administration of a calcium-deficient diet. Therefore, the ELISA developed in this study could be used for measuring bone resorption in the mouse model.

Association of Functional Microsatellites in the Human Type I Collagen α2 Chain (COL1A2) Gene with Systemic Sclerosis

Systemic sclerosis (SSc) or scleroderma is a generalized disorder of connective tissue. The etiology is poorly understood; however, both genetic and environmental factors have been implicated. To investigate the disease-susceptible gene for SSc, we examined the association of the disease with a gene (COL1A2) for type I collagen, which accumulates excessively in the affected organs. The COL1A2 gene containing a specific combination of the two dinucleotide repeats, repeat-haplotype, is involved in the regulation of gene expression. Homozygotes for a 5′-(CA)13CGCACA(CG)6(CA)8 -(GT)12 -3′ were found with significantly higher frequency (P = 0.029, relative risk, RR > 6.93) in SSc patients than in controls, and association was prominent (P = 0.0042, RR > 32.0) in the male patients positive for SSc-specific antinuclear antibodies (ANAs). This repeat-haplotype showed the highest stimulative activity for the transcription of the COL1A2 promoter among the reporter gene constructs tested. The results indicate that a portion of the patients having a specific dinucleotide repeat-haplotype homozygously and expressing the ANAs have a significantly higher risk for SSc than those individuals with other combinations of the repeat-haplotypes.

Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin.

The substrate specificity of porcine pepsin has been altered by site-directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro, Yaa is Glu. Leu, Gln or Lys, Nph is p-nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two- to fourfold higher kcat/Km, compared with wild-type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild-type pepsin cleaved a peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen alpha1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met cleaved site Ser-Phe at a rate 20-fold higher than the wild-type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu-Ser (similar to cathepsin D) and at a rate 23-fold higher than the wild-type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.

Alport Syndrome with Diffuse Leiomyomatosis: When and When Not?

Alport Syndrome with Diffuse Leiomyomatosis: When and When Not?

Different Behaviors of Serum and Urinary CrossLaps ELISA in the Assessment of Bone Resorption in Healthy Girls

Many fragments derived from collagen I degradation come to serum during bone resorption. The characterization of these fragments and the development of assays that can measure them are the basis of most of the biochemical markers of bone resorption. One of these fragments consists of two cross-linked amino acid sequences derived from the C-terminal telopeptide region of type I collagen α1 chains. Fragments containing the sequence EKAH-DGGR (CTX), with a DG site being either nonisomerized (α-CTX) or β-isomerized (β-CTX) were identified. Pyridinoline, deoxypyridinoline, and other cross-links linked both telopeptides (Fig. 1⇓ ) (1). Recent reports have suggested that the proportions of nonisomerized and β-isomerized fragments depend on bone age (1). There are two available assays that can measure the β-isomer form of the CTX epitope: urinary CrossLapsTM ELISA (2), and Serum CrossLaps One Step ELISA (3), both from Osteometer (Denmark). The urinary assay is a polyclonal (rabbit) antibody assay for which the minimal requirement for detection is the presence of …

A Patient with Ehlers-Danlos Syndrome Type VI Is Homozygous for a Premature Termination Codon in Exon 14 of the Lysyl Hydroxylase 1 Gene

In the present study, we have characterized a patient with Ehlers-Danlos syndrome type VI (EDS VI) as homozygous for a pathogenetic mutation in the lysyl hydroxylase 1 (LH1) gene. This mutant allele contributes to very low levels of LH1 mRNA and severely diminished LH activity in his skin fibroblasts. The reduced hydroxylysine content of collagen was reflected in the increased electrophoretic mobility of the type I collagen α1 and α2 chains precipitated from cell and media samples of cultured patient fibroblasts. The homozygous mutation, a single base change of C1557→ G which would convert a codon for tyrosine (TAC) at residue 511 to a stop codon (TAG) in exon 14 of the LH1 gene, was identified in full-length cDNAs for LH1 amplified from the patient's fibroblasts. We have demonstrated that the low level of LH activity measured in his fibroblasts may result from a minor processing pathway in which an in-frame skipping of exon 14 containing the mutation restores partial function of the enzyme. The mutation was confirmed in both alleles in genomic DNA from the proband and by the maternal inheritance of this mutation. The father's DNA was unavailable for analysis. The autosomal recessive nature of EDS VI was verified by the fact that the mother, who has one mutated and one normal allele, is clinically unaffected by this disorder. This mutation, which has been previously observed in another unrelated compound heterozygous patient, may prove to be a more widespread mutation for EDS VI.

Characterization of immortalized rat hepatic stellate cell lines transfected with SV40 T antigen

Hepatic stellate cells (HSCs) play an important role in fibrogenesis of the liver through their activity of biosynthesizing a widely variety of extracellular matrix proteins. However, in vitro experiments using primary cultured HSCs are limited, because HSCs in primary culture tend to dedifferentiate quickly. We recently established several HSC lines by transfection with SV 40 large T antigen (SV40 TAg) without any culture crisis. The cell lines have both morphological and functional characteristics of HSCs. Immunohistochemically, these cell lines showed positive staining of SV 40 TAg in nuclei and desmin and type IV collagen in cytoplasms. Cell lines also had messenger RNA expressions of type I collagen α1 chain, α-smooth muscle actin, hepatocyte growth factor, transforming growth factor-β 1. These cell lines would be useful for in vitro studies in regard to the mechanism of differentiation or transformation of rat HSCs.

Binding of proline- and hydroxyproline-containing peptides and proteins to the capillary wall

Sticking of peptides (as demonstrated by peak distortion) containing a high proportion of glycine and proline residues to the capillary wall was investigated. At acid pH where the carboxy terminal proline (e.g. Gly–Pro) is protonated, distinct sticking of these peptides occurred. On the contrary, the Pro–Gly peptide yielded a well shaped peak. At alkaline pH, where the situation was reversed, the peptide sticking most to the wall was Gly–Gly, while the behaviour of the dipeptides possessing N- or C-terminal proline did not suffer from sticking to the wall. It was concluded that the separation of simple proline- and glycine-containing dipeptides can be partly optimized by manipulating the pH of the background electrolyte; particularly if cetyltrimethylammonium bromide, methanol or acetonitrile is added to the run buffer (reversed polarity mode with cetyltrimethylammonium bromide). However, all attempts to resolve more complex mixtures of proline- (and hydroxyproline-) containing di- and tripeptides failed; addition of an organic modifier, i.e. methanol and acetonitrile, hexylamine, triethylamine and cetyltrimethylammonium bromide was unsuccessful. Such separations can be materialized by using simple 25 mmol/l phosphate buffer at pH 10.5 provided that the sample is dissolved in (aqueous) 17.5 mmol/l Brij or 33 mmol/l sodium dodecyl sulfate. These systems are also applicable to large polypeptides rich in proline, hydroxyproline and glycine: typically parent collagen α-chains, their dimers, trimers and higher polymers were successfully separated.

Adhesive Domains in the Collagen III Fragment α1(III)CB4 that Support α2β1- and von Willebrand Factor-mediated Platelet Adhesion under Flow Conditions

Seven overlapping peptides derived from the bovine alpha1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin alpha2beta1-recognition site has been assigned within this fragment to residues 522-528 of the collagen alpha1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both alpha2beta1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solid-phase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the alpha1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the alpha2 subunit of integrin alpha2beta1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the alpha2beta1-recognition site in this locality in alpha1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the alpha1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of alpha2beta1-, vWF- and rLAPP-binding sites all in close proximity in alpha1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

Clinical evaluation of the Serum CrossLaps One Step ELISA, a new assay measuring the serum concentration of bone-derived degradation products of type I collagen C-telopeptides

The Serum CrossLaps One Step ELISA is a sandwich assay using two monoclonal antibodies specific for a beta-aspartate form of the epitope EKAHDGGR derived from the carboxy-terminal telopeptide region of type I collagen alpha1-chain. Our objective was to assess the clinical value of the Serum CrossLaps assay for monitoring antiresorptive therapy in osteoporosis treatment. Samples obtained from postmenopausal women treated with different doses of cyclic or continuous hormone replacement therapy (HRT) with an estrogen analog (tibolone) or with a bisphosphonate (ibandronate) were measured in the Serum CrossLaps One Step ELISA at baseline and at various time points during therapy. The corresponding urine samples were measured in the urine CrossLaps ELISA and corrected for creatinine excretion. The serum CrossLaps measurements and corresponding urinary CrossLaps measurements were highly correlated (r >0.8 for all studies). The serum and urine CrossLaps measurements showed a significant decrease among the women treated with clinically relevant doses of either of the antiresorptive agents. Furthermore, the annual percentage change in bone mineral density (BMD) correlated with the measured changes in CrossLaps concentration. The serum CrossLaps assay showed a specificity of 83-100% and a sensitivity of 59-83% for assessing BMD changes. The corresponding values for the creatinine-corrected urinary measurements were 83-92% specificity and 68-79% sensitivity. We conclude that performance of the convenient Serum CrossLaps One Step ELISA is at least equivalent to that of the urine text for follow up of antiresorptive treatment in osteoporosis. Further studies are needed to optimize its use in this and other clinical applications.

Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N- 1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure

CB2, a CNBr peptide of 36 residues from type I collagen α1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg 9 showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons ( 1 J NH=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (Δδ/Δ T of −3.6 ppb/°C) suggest that the H atoms of the protonated guanidinium moiety of Arg 9 in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354–7359), similar to CB2, our data could indicate that the guanidinium group of Arg 9 form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the N ϵ hydrogen, leaving the four N η hydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric α1(I) CB2 triple helix. The behaviour of Arg 9 has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17°C.

Expression of mRNA for type IV collagen alpha1, alpha5 and alpha6 chains by cultured dermal fibroblasts from patients with X-linked Alport syndrome.

COL4A5 mutations causing X-linked Alport syndrome (XLAS) are frequently associated with absence of the alpha3, alpha4,alpha5 and alpha6 chains of type IV collagen from basement membranes and increased amounts of the alpha1(IV) and alpha2(IV) chains in glomerular basement membrane. Although many COL4A5 mutations have been described in XLAS, the mechanisms by which these mutations influence the basement membrane appearance of chains other than alpha5(IV) remain poorly understood. In this study, we used dermal fibroblasts from eight normal individuals and nine males with XLAS to test the hypotheses that COL4A5 mutations increase transcription of COL4A1 and suppress transcription of COL4A6. Ribonuclease protection assays revealed that alpha1(IV), alpha5(IV) and alpha6(IV) transcripts were expressed in cultures of dermal fibroblasts. The mRNA levels for alpha1(IV) in eight of nine patients with XLAS were not increased compared to controls; one patient with a large COL4A5 deletion showed significant elevation of alpha1(IV) mRNA levels. No differences in steady-state mRNA levels for alpha6(IV) were found when XLAS fibroblasts were compared with controls, even though little or no alpha6(IV) protein was detectable at the dermal-epidermal junction by immunofluorescence study. This finding suggests that post-transcriptional events account for the absence of alpha6(IV) in the Alport dermal-epidermal junction.

Epithelial vs Mesenchymal Contribution to the Extracellular Matrix in the Human Intestine

The basement membrane (BM) underlying the epithelium of the intestine is generally believed to be of both epithelial and mesenchymal origin but the exact contribution of each tissue has not been directly examined in the human. In this study, we have used a newly described procedure to dissociate the human intestine into pure epithelial and corresponding mesenchymal fractions. Northern blot and RT-PCR analyses of the fractions for the presence of transcripts encoding extracellular matrix molecules revealed that the epithelium produces the formal BM molecules such as the α1, α2, and β1 chains of laminin-1 and laminin-2 and the α5(IV) and α6(IV) chains of collagen as well as fibronectin, a BM-associated molecule. Interestingly, the α1(IV) chain of collagen, which associates with the α2(IV) chain to form the main BM collagen network, as well as tenascin-C and decorin, two BM-associated molecules, was found to be exclusively of mesenchymal origin. Taken together, these data support the concept that in the human, as in experimental animals, the intestinal BM is composed of components produced from both the epithelium and the mesenchyme.

Complete Exon-Intron Organization of the Human Gene for the α1 Chain of Type XV Collagen (COL15A1) and Comparison with the Homologous Col18a1 Gene

The human gene for the alpha1 chain of type XV collagen (COL15A1) is about 145 kilobases in size and contains 42 exons. The promoter is characterized by the lack of a TATAA motif and the presence of several Sp1 binding sites, some of which appeared to be functional in transfected HeLa cells. Comparison with Col18a1, which encodes the alpha1(XVIII) collagen chain homologous with alpha1(XV), indicates marked structural homology spread throughout the two genes. The mouse Col18a1 contains one exon more than COL15A1, due to the fact that COL15A1 lacks sequences corresponding to exon 3 of Col18a1, which encodes a cysteine-rich sequence motif. Twenty-five of the exons of the two genes are almost identical in size, six of them contain conserved split codons, and the locations of the respective exon-intron junctions are identical or almost identical in the two genes. The homologous exons include the closely adjacent first pair of exons and the exons encoding a thrombospondin-1 homology found in the N-terminal noncollagenous domain 1, which are followed by the most variable part of the two genes, covering the C-terminal half of their noncollagenous domain 1 and the beginning of the collagenous portion, after which most of the exons are homologous. The lengths of the introns are not similar in these genes, with two exceptions, namely the first intron, which is very short, less than 100 base pairs, and the second intron, which is very large, about 50 kilobases, in both genes. It can be concluded that COL15A1 and Col18a1 are derived from a common ancestor.

Estradiol reverses TGF-beta1-stimulated type IV collagen gene transcription in murine mesangial cells.

We have previously shown that estradiol suppresses types I and IV collagen synthesis by mesangial cells grown in the presence of serum. In the present study, we examined the interaction between estradiol and transforming growth factor-beta (TGF-beta) on collagen IV synthesis. In a luciferase reporter gene construct containing the type IV collagen promoter and 1-chain regulatory sequences, we found that TGF-beta1 (2 ng/ml) stimulated alpha1-collagen IV gene transcription in serum-free media (140.5 +/- 6.2 relative luciferase units, expressed as a percent of control untreated cells, P < 0.001). Estradiol reversed the stimulatory effects of TGF-beta1 on reporter gene transcription in a dose-dependent manner [for 2.5 x 10(-9) M, 114.2 +/- 0.2, P < 0.002 vs. TGF-beta1; for 10(-7) M, 89.5 +/- 4.0, P < 0.001 vs. TGF-beta1 and P = not significant (NS) vs. control]. Using immunoprecipitation techniques, we found that estradiol (10(-7) M) reversed TGF-beta1-stimulated type IV collagen synthesis (175.3 +/- 14.7 vs. 111.6 +/- 7.1, expressed as a percent of control untreated cells, P < 0.001) but did not affect TGF-beta1-stimulated type I collagen synthesis (166.9 +/- 18.8 vs. 162.2 +/- 16.2, P = NS). These results were confirmed with Western blotting. Nuclear extracts from mesangial cells treated with TGF-beta1 showed increased binding to a Sp1 consensus binding sequence oligonucleotide and to an Sp1 binding site in the collagen IV promoter. Estradiol reversed this enhanced binding. These data suggest that estradiol antagonizes TGF-beta1-stimulated type IV collagen synthesis at a transcriptional level and that this effect may be mediated by interactions with the transcription factor Sp1.

Stretch-Induced Collagen Synthesis in Cultured Smooth Muscle Cells from Rabbit Aortic Media and a Possible Involvement of Angiotensin II and Transforming Growth Factor-β

Mechanical strain reportedly stimulates the synthesis of collagen in vascular smooth muscle cells (SMCs). The present study was designed to investigate a possible involvement of angiotensin II (Ang II) and transforming growth factor (TGF)-β in stretch-induced collagen synthesis of cultured SMCs derived from the rabbit aortic media. SMCs were cyclically stretched at a rate of 10% elongation and 30 cycles/min for 24 h using the Flexercell^® strain unit (Flexcell International Corp., McKeesport, Pa.). A two-fold increase in collagen synthesis and a concurrent increase in total protein synthesis were noted in stretched SMCs. Concentration of immunoreactive Ang II in the conditioned medium was elevated under the mechanical strain. Stretch-induced collagen and total protein synthesis were inhibited by either a selective antagonist to Ang II (saralasin), an angiotensin I-converting enzyme inhibitor (captopril) or an antisense oligonucleotide for angiotensinogen mRNA. An elevated secretion of TGF-β, both active and latent forms, was found in the medium of stretched SMCs. Saralasin inhibited the stretch-induced secretion of TGF-β from SMCs. Stretch-induced collagen and total protein synthesis was further inhibited by either an anti-TGF-β1 neutralizing antibody or an adenovirus-mediated transfer of a truncated TGF-β type II receptor. Elevated expression of collagen α1(III) chain and TGF-β1 mRNAs, and its reversal by saralasin were also demonstrated in stretched SMCs. Results indicate that the stretch-induced collagen and total protein synthesis appears to be mediated via an autocrine-paracrine mechanism of Ang II and TGF-β released from SMCs.

Expression of type VI collagen in the developing mouse heart

During development, the embryonic atrioventricular (AV) endocardial cushions undergo a morphogenic process to form mature valve leaflets and the membranous septa in the heart. Several extracellular matrix (ECM) proteins are expressed in the developing AV endocardial cushions, but it remains to be established if any specific ECM proteins are necessary for normal cushion morphogenesis. Abnormal development of the cardiac AV valves is a frequent cause of congenital heart defects, particularly in infants with trisomy 21 (Down syndrome). The genes encoding the α1 and α2 chains of type VI collagen are located on human chromosome 21 within the region thought to be critical for congenital heart defects in trisomy 21 infants. This suggests that the type VI collagen α1(VI) and α2(VI) chains may be important in normal AV valve morphogenesis. As a first step in understanding the role of type VI collagen in valve development, the authors examined the normal spatial and temporal expression patterns of mRNA and protein for type VI collagen in the embryonic mouse heart. Ribonuclease protection assay analysis demonstrates cardiac expression of the type VI collagen for α1(VI), α2(VI), and α3(VI) transcripts beginning at embryonic days 11–11.5 of mouse development. In situ hybridization studies demonstrate a coordinated pattern of cardiac expression within the AV valves for each type VI collagen chain from embryonic day 11.5 through the neonatal period. Immunohistochemical studies confirm a concentrated type VI collagen localization pattern in the endocardial cushions from the earliest stages of valve development through the neonatal period. These data indicate that type VI collagen is expressed in the developing AV canal in a pattern consistent with cushion tissue mesenchymal cell migration and proliferation, and suggest that type VI collagen plays a role in the morphogenesis of the developing cardiac AV endocardial cushions into the valve leaflets and membranous septa of the heart. Dev. Dyn. 1998;211:248–255. © 1998 Wiley-Liss, Inc.

Expression of type VI collagen in the developing mouse heart.

During development, the embryonic atrioventricular (AV) endocardial cushions undergo a morphogenic process to form mature valve leaflets and the membranous septa in the heart. Several extracellular matrix (ECM) proteins are expressed in the developing AV endocardial cushions, but it remains to be established if any specific ECM proteins are necessary for normal cushion morphogenesis. Abnormal development of the cardiac AV valves is a frequent cause of congenital heart defects, particularly in infants with trisomy 21 (Down syndrome). The genes encoding the alpha1 and alpha2 chains of type VI collagen are located on human chromosome 21 within the region thought to be critical for congenital heart defects in trisomy 21 infants. This suggests that the type VI collagen alpha1(VI) and alpha2(VI) chains may be important in normal AV valve morphogenesis. As a first step in understanding the role of type VI collagen in valve development, the authors examined the normal spatial and temporal expression patterns of mRNA and protein for type VI collagen in the embryonic mouse heart. Ribonuclease protection assay analysis demonstrates cardiac expression of the type VI collagen for alpha1(VI), alpha2(VI), and alpha3(VI) transcripts beginning at embryonic days 11-11.5 of mouse development. In situ hybridization studies demonstrate a coordinated pattern of cardiac expression within the AV valves for each type VI collagen chain from embryonic day 11.5 through the neonatal period. Immunohistochemical studies confirm a concentrated type VI collagen localization pattern in the endocardial cushions from the earliest stages of valve development through the neonatal period. These data indicate that type VI collagen is expressed in the developing AV canal in a pattern consistent with cushion tissue mesenchymal cell migration and proliferation, and suggest that type VI collagen plays a role in the morphogenesis of the developing cardiac AV endocardial cushions into the valve leaflets and membranous septa of the heart.