Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys.

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.

Collagen IV of basement membranes: III. Chloride pressure is a primordial innovation that drives and maintains the assembly of scaffolds.

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.

Collagen IV of basement membranes: I. Origin and diversification of COL4 genes enabling animal evolution.

Collagen IV is a primordial component of basement membranes, a specialized form of extracellular matrix that enabled multi-cellular epithelial tissues. In mammals, collagen IV assembles from a family of six α-chains (α1 to α6), encoded by six genes (COL4A1 to COL4A6), into three distinct scaffolds: the α121, the α345 and a mixed scaffold containing both α121 and α565. The six mammalian COL4A genes occur in pairs that occur in a head-to-head arrangement on three distinct chromosomes. In Alport syndrome, variants in the COL4A3, 4 or 5 genes cause either loss or defective assembly of the collagen IV α345 scaffold which results in a dysfunctional glomerular basement membrane, proteinuria and progression to renal failure in millions of people worldwide. Here, we determine the evolutionary emergence and diversification of the COL4A genes using comparative genomics and biochemical analyses. Using syntenic relationships to genes closely linked to the COL4A genes, we determine that the COL4A3 and COL4A4 gene pair appeared in cyclostomes (hagfish and lampreys) while the COL4A5 and COL4A6 gene pair emerged in gnathostomes, jawed vertebrates. The more basal chordate species, lancelets and tunicates, do not have discrete kidneys and have a single COL4A gene pair, though often with single isolated COL4 genes similar to those found in C elegans . Remarkably, while the six COL4A genes are conserved in vertebrates, amphibians have lost the COL4A3 and COL4A4 genes. Our findings of the evolutionary emergence of these genes, together with the amphibian double-knockout, opens an experimental window to gain insights into functionality of the Col IV α345 scaffold.

Collagen IV and basement membrane at the evolutionary dawn of metazoan tissues.

The role of the cellular microenvironment in enabling metazoan tissue genesis remains obscure. Ctenophora has recently emerged as one of the earliest-branching extant animal phyla, providing a unique opportunity to explore the evolutionary role of the cellular microenvironment in tissue genesis. Here, we characterized the extracellular matrix (ECM), with a focus on collagen IV and its variant, spongin short-chain collagens, of non-bilaterian animal phyla. We identified basement membrane (BM) and collagen IV in Ctenophora, and show that the structural and genomic features of collagen IV are homologous to those of non-bilaterian animal phyla and Bilateria. Yet, ctenophore features are more diverse and distinct, expressing up to twenty genes compared to six in vertebrates. Moreover, collagen IV is absent in unicellular sister-groups. Collectively, we conclude that collagen IV and its variant, spongin, are primordial components of the extracellular microenvironment, and as a component of BM, collagen IV enabled the assembly of a fundamental architectural unit for multicellular tissue genesis.

A unique covalent bond in basement membrane is a primordial innovation for tissue evolution.

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad-a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids-is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Prospective randomized study of carotid endarterectomy with polytetrafluoroethylene versus collagen-impregnated Dacron (Hemashield) patching: perioperative (30-day) results.

PURPOSE: Several studies have reported that carotid endarterectomy (CEA) with patch angioplasty has results that are superior to primary closure. Polytetrafluoroethylene (PTFE) patching has been shown to have results comparable with autogenous vein patching; however, it requires a prolonged hemostasis time. Therefore, many surgeons are using collagen-impregnated Dacron patching (Hemashield [HP]). This study is the first prospective randomized trial comparing CEA with PTFE patching versus HP patching. METHODS: Two hundred CEAs were randomized into two groups, 100 PTFE and 100 HP patching. All patients underwent immediate postoperative and 1-month postoperative color duplex ultrasound scanning studies. Demographic and clinical characteristics were similar in both groups, including the mean operative diameter of the internal carotid artery. RESULTS: The perioperative stroke rates were 0% for PTFE, versus 7% for HP (4 major and 3 minor strokes, P =.02). The combined perioperative stroke and transient ischemic attack rates were 3% for PTFE, versus 12% for HP (P =.047). The operative mortality rate for PTFE was 0%, versus 2% for HP (P =.477). Five perioperative carotid thromboses were noted in patients undergoing HP patching, versus none in patients undergoing PTFE patching (P =.07). After 1 month of follow-up, 2% of patients in the PTFE group had a 50% or more restenosis, versus 12% of patients in the HP group (P =.013). The mean operative time for PTFE patching was 119 minutes, versus 113 minutes for HP patching (P =.081). The mean hemostasis time was significantly higher for PTFE patching than for HP patching, 14.4 versus 3.4 minutes (P <.001). CONCLUSION: CEA with HP patching had a higher incidence of perioperative strokes, carotid thrombosis, and 50% or more early restenosis than CEA with PTFE patching. However, the mean hemostasis time was higher for PTFE patching than for HP patching.