Dedifferentiated cardiomyocytes from chronic hibernating myocardium are ischemia-tolerant

Left ventricular biopsies from 21 patients with clinically diagnosed chronic hibernating myocardium (CHM) were examined by light- and electron microscopy. A mean of 27% of cardiomyocytes were structurally altered and were characterized as hibernating, because of reduced amount of myofibrils and increased glycogen content. Electron microscopy of these cells showed reduction of T-tubules and numerous small mitochondria, but few changes associated with degeneration, acute ischemia or apoptosis. The structural changes found in CHM are reminiscent of dedifferentiation rather than degeneration. The expression patterns of some structural proteins show resemblance with those in embryonic cardiomyocytes.Histochemically, mitochondrial NADH-oxidase and proton translocating ATPase activities were absent, whereas cytochrome c activity was present. Intracellular calcium distribution indicated normally bound sarcolemmal calcium and absence of excess mitochondrial calcium accumulation. Nuclear chromatin ranged from normal to dispersed with only a few nuclei that were clumped. These results suggest that cardiomyocytes from human CHM hearts are structurally altered, but viable, and lack morphologic and cytochemical characteristics suggestive of apoptosis or acute ischemia.     

Increased glycolysis as protective adaptation of energy depleted,degenerating human hibernating myocardium

In the current study on human hibernating myocardium (HHM), we tested the hypothesis that increased glycolysis might exert a positive effect during a supply-demand balance situation by augmentation of myocardial energy formation. In 14 patients HHM was preoperatively detected by clinical methods and validated by the recovery of contractile function three months following revascularization. During open-heart surgery, transmural biopsies were removed from the hibernating areas and analyzed using biochemical and morphologic methods. Metabolite contents were normalized for the degree of fibrosis (control: 9.8 ± 0.5%, HHM 28.1 ± 3.0%; p < 0.05), providing values for cardiomyocytes only. In energy depleted HHM, severe intracellular degeneration, glycogen accumulation and myocyte loss were found. Elevated lactate levels (2.22 ± 0.26 vs. 25.38 ± 3.53 mol/wet wt, p < 0.001) were indicative of an increased anaerobic glycolytic flux. In conclusion the presence of abundant intracellular glycogen and an increased anaerobic glycolysis in HHM is indicative of a protective adaptation of this myocardium, which might balance energy deficit and may limit structural damage.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

Reduced sarcoplasmic reticulum Ca2+-ATPase activity and dephosphorylated phospholamban contribute to contractile dysfunction in human hibernating myocardium

Human hibernating myocardium (HHM) is characterized by reversible contractile dysfunction during chronic ischemia. A disturbed calcium-homeostasis is a decisive factor for reduced functional capacity in heart diseases. We therefore investigated calcium-handling proteins in HHM. In 12 patients suffering from multi-vessel coronary artery disease and contractile dysfunction with indication for bypass surgery, HHM was detected preoperatively by thallium scintigraphy, radionuclide ventriculography and dobutamine echocardiography. Transmural biopsies of these regions were taken and analyzed by immunohistochemistry and electron microscopy. Furthermore, SR-calcium ATPase (SERCA2a), phospholamban (PLN), the phosphorylated forms of PLN (PLN-Ser16, PLN-Thr17) as well as sodium-calcium exchanger (NCX) and ryanodine receptor (RyR2) were investigated by RT-PCR and Western-blotting. Additionally, SERCA2a activity was measured by an enzyme-coupled assay. In all patients complete functional recovery could be documented 3 months after revascularization by repeating all preoperative investigations. In HHM maximal SERCA2a activity was significantly reduced (HHM: 424.5± 33.9, control: 609.0± 48.5 nmol ATP mg protein⁻¹ min⁻¹, p≤ 0.05), whereas SERCA2a protein levels were unchanged. mRNA levels (HHM: 1.36± 0.08 vs. control: 0.78± 0.04, p≤ 0.05) and protein amount (HHM:1.67± 0.14 vs. control: 1.00± 0.04, p≤ 0.05) of PLN (A1) were increased resulting in an increased PLN:SERCA2a-ratio. PLN-Ser16 (HHM: 0.60± 0.08 vs. control: 1.00± 0.11, p≤ 0.05) and PLN-Thr17 (HHM: 0.63± 0.11 vs. control: 1.00± 0.06, p≤ 0.05) phosphorylation was significantly decreased. RyR2 and NCX showed no significant alteration. In HHM a decreased activity of SERCA2a due to an impaired phosphorylation of PLN contributes to contractile dysfunction. The increase in the relative ratio of PLN/SERCA2a leads to a decreased calcium affinity of SERCA2a.     

Hibernating myocardium: Its pathophysiology and clinical role

Myocardial hibernation, as first defined by Rahimtoola, is a state of chronic contractile dysfunction in patients with coronary artery disease which is fully reversible upon reperfusion. Clinical conditions consistent with the existence of myocardial hibernation include unstable and stable angina, myocardial infarction heart failure, and anomalous origin of coronary arteries. The mechanisms of hibernation are not known. Morphological alterations have been described in the hibernating area of patients, but these information are strongly affected by the diagnostic criteria utilized to screen patients. It has been postulated that hibernation is an adaptive phenomenon occurring during ischemia. In this context, downregulation of contraction is not regarded as a consequence of energetic deficit, but as a regulatory event aimed at reducing energy expenditure, thereby maintaining integrity and viability. Thus, hibernation might bear a relationship to the phenomenon of low-flow perfusion-contraction matching, or repetitive stunning or preconditioning. Clear-cut evidence for the mechanism of hibernation in the clinical setting seems likely to remain elusive, because of the nature of the studies needed to document it. Current experimental evidence supports the view that hibernation, stunning, preconditioning, or their coexistence can be responsible for regional myocardial contractile dysfunction which is reversible upon reperfusion. These are all adaptive and protective phenomena independent of their terminology and strict definitions and do not always apply to the extremely complex situation of myocardial ischemia in man.     

Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in three-dimensional cultures of rat aorta: A comparative study of angiogenesis in Matrigel,collagen, fibrin,and plasma clot

Summary Rings of rat aorta cultured in Matrigel, a reconstituted gel composed of basement membrane molecules, gave rise to three-dimensional networks composed of solid cellular cords and occasional microvessels with slitlike lumina. Immunohistochemical and ultrastructural studies showed that the solid cords were composed of endothelial sprouts surrounded by nonendothelial mesenchymal cells. The angiogenic response of the aortic rings in Matrigel was compared to that obtained in interstitial collagen, fibrin, or plasma clot. Morphometric analysis demonstrated that the mean luminal area of the microvascular sprouts and channels was significantly smaller in Matrigel than in collagen, fibrin, or plasma clot. The percentage of patent microvessels in Matrigel was also markedly reduced. Autoradiographic studies of³H-thymidine-labeled cultures showed reduced DNA synthesis by developing microvessels in Matrigel. The overall number of solid endothelial cords and microvessels was lower in Matrigel than in fibrin or plasma clot. A mixed cell population isolated from Matrigel cultures formed a monolayer in collagen or fibrin-coated dishes but rapidly reorganized into a polygonal network when plated on Matrigel. The observation that gels composed of basement membrane molecules modulate the canalization, proliferation, and organization into networks of vasoformative endothelial cells in three-dimensional cultures supports the hypothesis that the basement membrane is a potent regulator of microvascular growth and morphogenesis. This work was supported by grants from the W. W. Smith Charitable Trust and grants CA14137 and HL43392 from the National Institutes of Health, Bethesda, MD.     

Basement membranes in development and disease

Basement membranes (BMs) are specializations of the extracellular matrix that act as key mediators of development and disease. Their sheet like protein matrices typically serve to separate epithelial or endothelial cell layers from underlying mesenchymal tissues, providing both a biophysical support to overlying tissue as well as a hub to promote and regulate cell–cell and cell–protein interactions. In the latter context, the BM is increasingly being recognized as a mediator of growth factor interactions during development. In this review, we discuss recent findings regarding the structure of the BM and its roles in mediating the normal development of the embryo, and we examine congenital diseases affecting the BM which impact embryonic development and health in later life. Birth Defects Research (Part C) 90:8–31, 2010. © 2010 Wiley‐Liss, Inc.     

A self-perpetuating vicious cycle of tissue damage in human hibernating myocardium

Recently, we proposed the hypothesis that a vicious cycle exists in human hibernating myocardium (HM) between the progression of myocyte degeneration and the development of fibrosis [1]. We now investigated the pathomechanism of this cycle in more detail and established a correlation between the severity of the morphological changes and the degree of postoperative functional recovery of HM.HM was diagnosed by dobutamine echocardiography, thallium-201 scintigraphy and radionuclide ventriculography. Functional recovery was present at 3 months after coronary bypass surgery but remained unchanged at 15 months. Forty patients were subdivided into 2 groups: A with complete and B with incomplete recovery. Biopsies taken during surgery and studied by electron microscopy, immunocytochemistry, rt-PCR, and morphometry revealed myocyte degeneration and inflammatory and fibrinogenic changes in a widened interstitial space. We report here for the first time an upregulation of TGF-₁ evident by a 5-fold increase of fibroblasts and macrophages exhibiting a TGF-₁ content 3-fold larger than in control, and a > 3-fold increase in TGF-₁ mRNA by rt-PCR. The number of angiotensin converting enzyme (ACE) containing structures was increased (n/mm²: control - 11.4, A - 17.6, B - 19.2, control vs. A and B, p < 0.05). Fibrosis was more severe in group B than A or control (%: C - 10.1; A - 21.2; B - 40.6; p < 0.05). Capillary density was significantly reduced (n/mm²: C - 1152; A - 782; B - 579, p < 0.05) and intercapillary distance was widened (m: C - 29.5, A - 36.1, B - 43.3, p < 0.05). The number of CD 3 (n/mm²: C - 5.0; A - 9.6; B - 9.4, ns) and CD 68 positive cells (n/mm²: C - 37.2; A - 80.7; B - 55.0, C vs. A p < 0.05) was elevated in HM as compared to control indicating an inflammatory reaction. Cut-off points for functional recovery are fibrosis > 32%, capillary density < 660/mm² and intercapillary distance > 39.0 m.In HM a self-perpetuating vicious cycle of tissue alterations leads to progressive replacement fibrosis and continuous intracellular degeneration which should be interrupted by early revascularization.     

Characterization of a new tissue-engineered human skin equivalent with hair

Summary We designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.     

A basement membrane-associated glycoprotein from skeletal muscle

We have isolated a major glycoprotein that appears to be associated with rat skeletal muscle basement membrane. We determined that the glycoprotein was part of the muscle cell surface complex when we found it to be enriched in preparations of muscle ghosts. We isolate the glycoprotein from homogenized muscle preextracted with 4 M and 8 M urea. It elutes as a major component in the presence of 8 M urea/50 mM 2-mercaptoethanol. Its apparent molecular weight on sodium dodecyl sulfate gels is 130,000. Amino acid analysis indicates that it is not a collagen but that it does contain small amounts of hydroxyproline and hydroxylysine. There may be collagenous domains in the glycoprotein molecule, for it is cleaved into three fragments by purified bacterial collagenase. Immunoperoxidase staining confirms that the 130,000-dalton protein is localized at the surface of adult skeletal muscle cells. It is probably a general basement membrane-associated glycoprotein because we found material immunologically cross-reactive with the muscle glycoprotein in basement membrane regions of kidney, liver, brain, and small intestine. We have shown the glycoprotein to be distinct from fibronectin, laminin, and types I, III, IV, and V collagens in enzyme-linked immunosorbent assays.     

Biological activities of laminin

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.     

De-differentiation of primary human hepatocytes depends on the composition of specialized liver basement membrane

Despite the understanding of the importance of mitogen-activated protein (MAP) kinase activation in the stimulation of growth, little is known about the role of MAP kinase regulation during contact inhibited growth control. To investigate the role of the MAP kinase extracellular signal-regulated kinase (ERK) during the transition to a contact inhibited state, cultures of normal fibroblasts (BJ) were grown to different stages of confluency. The levels of MAP kinase phosphatase (MKP) expression and the amount of active ERK and MAP ERK kinase (MEK) in these cultures were assessed through western blot analysis and were compared to fibrosarcoma cell cultures (HT-1080), which lack contact inhibition. In normal fibroblasts, the amounts of active MEK and ERK decline at contact inhibition, concurrently with a rise in MKP-1, MKP-2, and MKP-3 protein levels. In contrast, fibrosarcoma cells appear to lack density-dependent regulation of the ERK pathway. Additionally, altering the redox environment of fibrosarcoma cells to a less reducing state, as seen during contact inhibition, results in increased MKP-1 expression. Taken together, these results suggest that the altered redox environment upon contact inhibition may contribute to the regulation of ERK inactivation by MKPs.     

A new technique for the isolation and purification of the basal lamina from insect tissues

A new technique for the isolation and purification of basal lamina from insect tissues using cell dissociation at pH 2 is described. Tissue incubation in these solutions results in the spontaneous detachment of cells from the basal lamina which can be collected free of any significant contamination by cellular components. Short lengths of plasma membrane which remain attached to the basal lamina can be removed by subsequent sonication or detergent treatment. Using Malpighian tubules as the primary test tissue, we have found that the procedure only requires a few minutes, works equally well on pooled tissue samples, individual tissue pieces or tissue subregions and involves no loss of basal lamina from the starting material.     

Expression of laminin chains by central neurons: analysis with gene and protein trapping techniques

Laminins exert numerous effects on neurons in vitro, but expression of laminin subunit genes by neurons in vivo remains controversial. To reexamine this issue, we generated mice from ES cells in which the laminin alpha1, alpha5, beta1, and gamma1 genes had been "trapped" by insertion of a histochemically detectable selectable marker, betageo (beta-galactosidase fused to neomycin phosphotransferase). The presence of laminin-betageo fusion proteins was assayed histochemically and immunochemically, revealing expression of laminin beta1 and gamma1 genes, but not alpha chain genes, by defined subsets of neurons in brain and retina. We also used the gene traps in a novel way to assay expression of endogenous laminin subunits, which were barely detectable by ordinary immunohistochemical methods. The trapping vector included a transmembrane domain that anchors proteins otherwise destined for secretion. Laminin alpha/beta/gamma heterotrimers are assembled intracellularly, and we show that the trapped laminin gamma1 fusion protein "co-trapped" endogenous beta1 intracellularly. The laminin gamma1 fusion was also able to co-trap transgene-derived alpha chains, but we detected no co-trapped endogenous alpha chains. The co-trapping method may be generally useful for identifying proteins or isolating protein complexes associated with trapped gene products.     

Building collagen IV smart scaffolds on the outside of cells

Collagen IV scaffolds assemble through an intricate pathway that begins intracellularly and is completed extracellularly. Multiple intracellular enzymes act in concert to assemble collagen IV protomers, the building blocks of collagen IV scaffolds. After being secreted from cells, protomers are activated to initiate oligomerization, forming insoluble networks that are structurally reinforced with covalent crosslinks. Within these networks, embedded binding sites along the length of the protomer lead to the “decoration” of collagen IV triple helix with numerous functional molecules. We refer to these networks as “smart” scaffolds, which as a component of the basement membrane enable the development and function of multicellular tissues in all animal phyla. In this review, we present key molecular mechanisms that drive the assembly of collagen IV smart scaffolds.     

β2 and γ3 laminins are critical cortical basement membrane components: Ablation of Lamb2 and Lamc3 genes disrupts cortical lamination and produces dysplasia

Cortical development is dependent on the timely production and migration of neurons from neurogenic sites to their mature positions. Mutations in several receptors for extracellular matrix (ECM) molecules and their downstream signaling cascades produce dysplasia in brain. Although mutation of a critical binding site in the gene that encodes the ECM molecule laminin γ1 (Lamc1) disrupts cortical lamination, the ECM ligand(s) for many ECM receptors have not been demonstrated directly in the cortex. Several isoforms of the heterotrimeric laminins, all containing the β2 and γ3 chain, have been isolated from the brain, suggesting they are important for CNS function. Here, we report that mice homozygous null for the laminin β2 and γ3 chains exhibit cortical laminar disorganization. Mice lacking both of these laminin chains exhibit hallmarks of human cobblestone lissencephaly (type II, nonclassical): they demonstrate severe laminar disruption; midline fusion; perturbation of Cajal‐Retzius cell distribution; altered radial glial cell morphology; and ectopic germinal zones. Surprisingly, heterozygous mice also exhibit laminar disruption of cortical neurons, albeit with lesser severity. In compound null mice, the pial basement membrane is fractured, and the distribution of a key laminin receptor, dystroglycan, is altered. These data suggest that β2 and γ3‐containing laminins play an important dose‐dependent role in development of the cortical pial basement membrane, which serves as an attachment site for Cajal‐Retzius and radial glial cells, thereby guiding neural development. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Phenotypic differences in subclones and long-term cultures of clonally derived rat bone cell lines

Previous studies with clonally derived populations of cells have shown that cells released from embryonic rat calvaria by enzymatic digestion are heterogeneous with respect to their hormone responsiveness, morphology, and production of matrix components [Aubin JE et al; J. Cell Biol 92:452, 1982]. Several of these clonal populations have been used to study the effects of long-term culture and inter- and intraclonal cell heterogeneity. During continuous subculture, marked changes in collagen synthesis were observed in two clonal populations. Both of these clones were originally responsive to parathyroid hormone (PTH) and synthesized primarily type I collagen with small amounts of type III and V collagens, although one clone (RCJ 3.2) had a fibroblastic morphology whereas the second clone (RCB 2.2) displayed a more polygonal shape. Following routine subculture over 3 yr, clone RCB 2.2 was found to synthesize exclusively alpha 1(I)-trimer and not other interstitial collagens. When the same cells were maintained at confluence for 1-2 wk, however, they also synthesized type III collagen. Whereas RCJ 3.2 did not show such dramatic changes in collagen synthesis after long-term subculture, two subclones derived from RCJ 3.2 were found to synthesize almost exclusively either type III collagen (RCJ 3.2.4.1) or type V collagen (RCJ 3.2.4.4). Immunocytochemical staining indicated that both subpopulations also produced type IV collagen, laminin, and basement membrane proteoglycan, proteins that are typically synthesized by epithelial cells. The differences in collagen expression by the various clonal cell populations were accompanied by qualitative and quantitative differences in other secreted proteins and differences in cell morphology. The results demonstrate both the inter- and intraclonal heterogeneity of connective tissue cells and their diverse potentiality with respect to extracellular matrix synthesis.     

Development of chicken embryos exposed to an intermittent horizontal sinusoidal 50 Hz magnetic field

The effects of intermittent exposure (2 h on/22 h off) to a 200 μT horizontal, sinusoidally oscillating (50 Hz) magnetic field were studied in 210 fertilized chicken eggs. Two hundred ten control eggs (sham-exposed) were incubated in the same chamber as the experimental eggs. Chick embryos were examined for developmental anomalies and maturity stage after 48 h of incubation. Immunohistochemical analysis of extracellular membrane components (laminin, fibronectin, and type IV collagen) were conducted on day 7 and histological examinations for malformations of brain, liver, and heart, on days 7, 12, and 18 of incubation. Furthermore, egg fertility and egg weights were evaluated on days 2, 7, 12, and 18. The investigation also measured the body weight of chickens for 90 days from hatching and included histological analysis of body organs. Each variable was investigated blind. Statistical comparison between exposed and sham-exposed values did not show significant differences in any of the variables investigated. Thus, it appears that the exposure of embryos to an intermittent 200 μT magnetic field at 50 Hz does not cause developmental anomalies, changes in maturity stage, alterations in distribution of extracellular membrane components, or malformations in the brain, liver, or heart. Moreover, there were no differences in body weight, morphology, or histology of central nervous system, liver, heart, or testis in 90-day-old chickens hatched from exposed in comparison to sham-exposed eggs. © 1996 Wiley-Liss, Inc.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.