INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BY VIRTUE of their unique ultrastructure, microvascular beds, composed of precapillary arterioles, capillaries, and postcapillary venules, are the principal sites of neovascularization (7, 24) and molecular exchange between the blood and the interstitial fluid (47). Because capillaries lack both a muscular wall and elaborate elastic subendothelial layers, the walls of these vessels consist only of the single layer of endothelial cells lining the lumen and a minimal subendothelial basal lamina (15). It is this basal lamina and surrounding periendothelial cells that provide the capillary with mechanical support (48). Microvascular beds such as the renal glomerular capillaries are especially dependent on the subendothelial basal lamina for tensile strength due to the high hydrostatic pressure experienced during the formation of plasma filtrate (10). Organized immediately subjacent to the capillary endothelium, the basal lamina consists of a meshwork of molecules that ensheath the endothelial and periendothelial cells (46). The intimate association among endothelium, basal lamina, and periendothelium facilitates rapid transport of molecules and provides a means of intercellular and intrasystemic communication.

Endothelial and periendothelial cells are the primary cellular constituents of the microvasculature. The continuous endothelial cell lining of the capillary lumen acts as the primary selective barrier, the tightness of which directly affects vessel wall permeability (43). Subsequently, capillary periendothelial cells ("pericytes") together with the basal lamina act as a molecular sieve that limits access to the interstitium (6). Pericytes and capillary endothelial cells act in concert to secrete and organize the basal lamina that forms such an integral component of capillary architecture (8, 13, 20, 23, 27, 30, 53, 55, 57).

Microvascular basal laminae, similar to those of larger muscular and elastic vessels, consist of a heterogeneous network of extracellular matrix proteoglycans and proteins. These molecules are integral components of the vascular architecture as well as regulators of cellular processes. Among the most thoroughly characterized capillary basal laminae are the renal glomerular basement membrane and the juxtaglomerular mesangial matrix (5, 14, 33, 40). A prominent constituent of these and other vascular matrices are the microfibrils, morphologically unique 10- to 12-nm diameter fibers (34) that occupy an amorphous, electron-lucent basal lamina region (7, 24). The necessity for properly assembled microfibrils is punctuated by the incidence of diseases in which a single allele of a microfibrillar component is mutated. Such disorders include Marfan's syndrome and congenital contractural arachnodactyly and are manifested as vascular and connective tissue abnormalities (12, 45). Microfibrils may also function as a repository for soluble effectors. For example, latent transforming growth factor- (TGF-) binding proteins are associated with microfibrils, where TGF- may be stored before activation (21, 38, 42). Likewise, the clotting factor thrombospondin has been localized to microfibrils in the vascular basement membrane, where it may mediate thrombogenesis in response to vascular injury (3). A subset of capillary basal lamina proteins bind select cell-surface integrins, thereby mediating cell-extracellular matrix adhesion (9-11). Integrins are heterodimeric, membrane-spanning receptors that serve as a molecular interface between the extra- and intracellular environment (2). Intracellularly, activation of signaling cascades in response to integrin ligation serves to modulate cell motility, gene expression, and differentiation state (29). Extracellularly, integrins impact adhesion to components of the extracellular milieu in response to cellular cues (29). It is precisely this biochemical complexity of capillary basal laminae that underlies their vital functions in structural support, cell-extracellular matrix interactions, and metabolite exchange.

The extracellular matrix also serves as a source of cues promoting angiogenesis and differentiation of the microvasculature. The spatiotemporal regulation of angiogenesis from existing capillary vessels is dependent on changes in the composition and organization of the extracellular matrix (51). The presence of appropriate growth factors and extracellular matrix constituents dictate both capillary sprouting and endothelial migration through the activation of discrete signaling cascades (50, 54). Until needed, some of these growth factors are maintained in an inactive state by sequestration within matrices. For example, following capillary injury fibroblast growth factor is immediately released from proteoglycans to potentiate angiogenesis and promote rapid wound healing (16). Capillary endothelial cells undergoing angiogenic migration secrete both extracellular matrix proteins and chemoattractants (51). These molecules in turn elicit the recruitment of interstitial fibroblasts and their subsequent differentiation into pericytes (49, 50). Given the developing scenario it is tempting to postulate that the cells comprising capillaries are expressly adapted to the synthesis of a unique basal lamina that supports both molecular exchange and angiogenesis.

Vascular transitions to and from the highly anastomosing capillary beds are delineated by gradual modulations in vessel architecture and cellular phenotype (19, 47). The diversity of endothelial and periendothelial cells along the vasculature is thought to represent a continuum of cellular differentiation states (49), as well as heterogeneity brought about by local demands (51). The recent discovery of proteins expressed exclusively by either venous or arterial endothelial cells, considered together with the morphological diversity of these cells, provides evidence of genetically determined distinctions between the cellular constituents of the circulation (1, 59). The interdependence between cellular differentiation and extracellular matrix constituents makes it plausible that the observed biochemical and morphological gradations are accompanied by changes in the molecular composition of microvascular basal laminae. Whereas the inventory of the microfibrillar and integrin-binding extracellular matrix proteins found in the basal laminae of capillaries and other vessels continues to grow, it is still incomplete. Furthermore, the identification of those molecular constituents selectively enriched in the microvascular wall remains a challenge. This study describes the first such candidate, a novel protein preferentially expressed in microvascular extracellular matrices.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents. Sodium dodecylsulfate, Triton X-100, Tween 20, AEBSF [4-(2-aminoethyl)benzenesulfonylfluoride, HCl], and Mowiol were obtained from Calbiochem (San Diego, CA). Affigel-10 activated resin was obtained from Bio-Rad (Hercules, CA). Horseradish peroxidase (HRP)-conjugated secondary antibodies and nitrocellulose membranes were supplied by Amersham (Arlington Heights, IL). Super Signal ECL was purchased from Pierce (Rockford, IL). FITC- and rhodamine-conjugated donkey anti-rabbit IgG, goat anti-mouse IgG, and alkaline phosphatase-conjugated goat anti-rabbit IgG were supplied by Jackson ImmunoResearch (West Grove, PA). Gold-conjugated (20 nm) goat anti-rabbit IgG, LR White embedding resin, uranyl acetate, lead citrate, and nickel grids were purchased from Ted Pella (Redding, CA). A mouse monoclonal antibody (A028) directed against an endothelial cell surface marker was obtained from Telios Pharmaceuticals (San Diego, CA) in the form of ascites. All other chemicals and reagents were purchased from Sigma (St. Louis, MO).

Isolation and characterization of cDNA. An oligo(dT)-primed -ZAP human placental cDNA library (3 × 10¹⁰ independent clones) was obtained from Stratagene (La Jolla, CA). The library was plated onto Escherichia coli XL-1 Blue (Stratagene) lawns and grown at 42°C for 3.5 h. Nitrocellulose filters impregnated with 10 mM isopropylthio--D-galactopyranoside were applied to the agar plates and incubated at 37°C for an additional 3.5 h. The filters were removed and washed in Tris-buffered saline (TBS, 50 mM Tris · HCl, pH 8.0, 200 mM NaCl), and nonspecific binding sites were blocked for 1 h with TBS-T (TBS, 0.1% Tween 20) containing 0.2% newborn calf serum. Antiserum directed against a unique peptide (AVWPTRTQGPLPSSLGK) or affinity-purified antipeptide antibodies (preadsorbed against E. coli lysate) were diluted in TBS-T containing 0.2% newborn calf serum and incubated with the membranes for 30 min at room temperature under continuous agitation. The membranes were washed five times with TBS-T (5 min each) at room temperature. After the washes, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG diluted in TBS-T containing 0.2% newborn calf serum for 30 min at room temperature. After being washed with TBS-T (five times, 5 min each), positive plaques were identified using nitroblue tetrazolium (0.05%) and bromo-chloro-indolylphosphate (0.015%) in TBS. Membranes were aligned with the agar plates and positive plaques were isolated by suspending core samples from the agar plates in 1 ml of 50 mM Tris · HCl, pH 7.5, containing 100 mM NaCl, 2 mM MgSO₄, 0.01% gelatin, and 3% chloroform. XL-1 Blue cells were grown to OD₆₀₀ = 1.0, mixed with isolated Bluescript phagemid stock and ExAssist helper phage (Stratagene). The cells were incubated for 15 min at 37°C, additional media were added to the infected cells, and the mixture was incubated at 37°C for an additional 2 h. Cells were pelleted by centrifugation for 15 min at 2,000 g. The supernatant was collected, heated at 70°C, and cleared by centrifugation for 15 min at 4,000 g. The supernatant containing filamentous phage was collected and used to infect E. coli strain SOLR cells (Stratagene). Isolated colonies were selected on ampicillin plates, and plasmids were isolated with the QIAprep Miniprep kit (Qiagen, Valencia, CA).

Plasmid inserts were sequenced using the GIBCO BRL Life Technologies (Gaithersburg, MD) cycle sequencing kit. Sequence analysis of all isolated clones resulted in the identification of 10 cDNA clones, 4 of which were identical (termed c4) and chosen for sequence analysis. Sequencing primers were supplied by Operon (Alameda, CA). Forward primers corresponded to T3 promotor sequences contained within pBluescript (5'-AATTAACCCTCACTAAAGGG-3') and bases 166-193, 375-394, and 444-466 of the c4 cDNA. Reverse primers corresponded to T7 promotor sequences contained within pBluescript (5'-GTAATACGACTCACTATAGGGC-3') and bases 1550-1567 of the c4 cDNA. PCR constructs encoding central portions of the c4 cDNA (either from bases 444-1567 or from bases 580-1176) were used to sequence these regions using the T3 and T7 primers (see above). Primers were end-labeled using T4 kinase with [-³³P]ATP (2-4 Ci/mol) (NEN Life Sciences Products, Boston, MA) (52). The dideoxy sequencing reactions were performed as per the manufacturer's directions. Sequencing gels were run on a Genomyx LR Programmable DNA sequencing apparatus (Genomyx, Fullerton, CA) maintained at 65°C. Sequence analyses were performed using the GCG software package (Wisconsin software package version 10, Genetics Computer Group, Madison, WI).

Northern blotting. A 1340-bp probe corresponding to the 5' region of cablin cDNA was labeled by random priming (52). The approximate size and tissue expression of cablin mRNA was determined by hybridization of the radioactive probe to a multiple tissue Northern blot (Clontech, Palo Alto, CA) loaded with 2 µg of poly(A)⁺ mRNA per lane using ExpressHyb solution (Clonetech) and following the manufacturer's recommended protocol. As a control for RNA loading, the same blot was subsequently probed with a 1200-bp probe for rat glyceraldehyde-3-phosphate dehydrogenase (18).

Recombinant protein expression and purification. The cablin cDNA insert was subcloned into pET21b (Novagen, Madison, WI) and used to express the encoded protein in E. coli strain BL-21/DE3 (Stratagene) (58). The detergent-insoluble material remaining after bacterial lysis was solubilized in 8 M urea for 1 h at room temperature and dialyzed successively against six changes of 50 mM Tris · HCl, pH 8.0, 50 mM NaCl, and 1 mM EDTA containing decreasing concentrations of urea. The dialysate was assayed for purity by SDS-PAGE followed by Coomassie stain, and the pure protein was diluted in phosphate-buffered saline (PBS) and injected (200 µg/injection) into rabbits for antibody production (Covance, Denver, PA). Freund's complete adjuvant was used for the primary injection, and Freund's incomplete adjuvant was used for all subsequent booster injections.

Affinity purification of antibodies. Recombinant protein was dialyzed against two changes of PBS and coupled to Affigel-10 resin according to manufacturer's directions. Immune rabbit serum was bound to the resin-bound antigen and specific antibodies eluted from the column using standard procedures (25).

Cell culture and extract preparation. Normal human aortic smooth muscle cells, human lung microvascular endothelial cells, and human umbilical cord vein endothelial cells and their defined media were purchased from Clonetics (San Diego, CA). Cells were seeded at 3,500 cells/cm² and allowed to grow to confluence over 3 days in a humidified atmosphere of 5% CO₂ at 37°C. Serum-free medium was added to confluent cultures and collected after 18 h. Medium from one 35-mm dish was concentrated 10-fold and mixed with SDS-PAGE sample buffer containing dithiothreitol. Cells were rinsed briefly in PBS, scraped from the dish, and pelleted by centrifugation at 700 g for 5 min. The cell pellet was resuspended in sample buffer, sonicated for 30 s, and boiled for 5 min before being loaded onto gels.

Preparation of tissue extracts. A 0.5-g sample of normal human kidney cortex was thawed from 80°C and minced on ice. The tissue was further homogenized in 15 mM Tris · HCl, pH 8.0, and 1 µM AEBSF with a Tissue Tearor (BioSpec Products, Bartlesville, OK) until completely liquefied. The homogenate was clarified by centrifugation for 10 min at 10,000 g at 4°C. The pellet fraction was resuspended in 5 ml of 10 mM Tris · HCl, pH 8.0, 2% SDS, 2% TX100, 10 mM EDTA, 0.5 M NaCl, 50 mM dithiothreitol, 1 µM AEBSF, and 1 µM CLAP (chymostatin, leupeptin, antipain, and pepstatin A), and incubated at 4°C for 8 h while being rocked. The extracted pellet was again subjected to centrifugation, and the resulting supernatant fraction was filtered through a 0.4-µm syringe filter, SDS-PAGE sample buffer was added, and the fraction was treated with 63 mM iodoacetamide (added as a solid) for 30 min at room temperature in the dark.

SDS-PAGE and Western blot analysis. Proteins were separated on 10% SDS-PAGE gels. After electrophoresis, gels were transferred to nitrocellulose membranes. Nonspecific binding sites on immunoblots were blocked by a 1-h incubation period at room temperature with 0.5% nonfat dried milk in TBS-T. Blots were probed with 1 µg/ml of primary antibody (in some cases preincubated with a 10-fold molar excess of immunogen) followed by 1 µg/ml of HRP-conjugated secondary antibodies. Bound HRP-conjugated antibodies were detected using SuperSignal ECL reagent. Control blots were probed with preimmune serum and never gave any signal.

Angiogenesis model. Hypoxia-induced neovascularization was incited by following established experimental procedures (56). Briefly, mice were placed in a hyperoxic environment of 75% oxygen from postnatal days 7-12. From postnatal days 12-17 mice were returned to atmospheric oxygen (a hypoxic environment relative to 75% oxygen), after which time they were euthanized. Eyes were enucleated, embedded in OCT compound, and 10-µm frozen sections cut for immunohistochemical analysis.

Immunofluorescence microscopy. Unfixed frozen tissue sections embedded in OCT were cut (5-10 µm in thickness) and collected on Probe-On Plus microscope slides (Fisher Scientific, Chicago, IL). Sections were blocked with PBS containing 0.2% cold water fish skin gelatin for 30 min at room temperature before incubation with antibodies. Sections were incubated with either affinity-purified antibodies, immune serum, or preimmune serum at a concentration of 10 µg/ml for 1 h at 37°C. Primary antibodies were detected with FITC- or rhodamine-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (20 µg/ml), and sections were mounted in Mowiol 4-88 or Vectashield-DAPI (Vector, Burlingame, CA). No signal was observed in sections stained with preimmune serum. Images were collected using an inverted Zeiss LSM 410 or Biorad MRC600, both equipped with He-Ne and Ar-Kr lasers.

Histological staining. Sections were stained with Mayer's hematoxylin and counter-stained with eosin (Sigma) according to manufacturer's instructions. Elastic fibers in bovine mitral valve wall were visualized by staining sections with a 1/10 aqueous solution of India ink for 3 min, followed by extensive rinsing with water.

Immunoelectron microscopy. A normal kidney from a Sprague-Dawley male rat was excised immediately after decapitation and chopped into 0.5-mm² pieces in 0.1 M sodium cacodylate, 2.5% glutaraldehyde, 2% paraformaldehyde, and incubated in this fixative for 2 h at room temperature. Reactive aldehyde groups were quenched with 50 mM ammonium chloride in 0.1 M sodium cacodylate for 30 min at room temperature. The tissue was then extracted for 30 min at room temperature with 0.1% saponin in 0.1 M sodium cacodylate then rinsed briefly in this buffer alone. The tissue was dehydrated by successive incubations (5 min each at room temperature) in 10%, 30%, and 50% ethanol followed by two incubations (30 min each at room temperature) in 70% ethanol. The minced pieces were embedded in LR White resin according to manufacturer's instructions. Sections (60- to 70-nm-thick) were cut from the polymerized blocks and collected on Formvar-coated nickel grids. Sections were immunolabelled by floating them upside down on drops of primary antibody diluted to 10 µg/ml in PBS containing 0.2% cold water fish skin gelatin for 1 h at room temperature, rinsing several times in PBS, and incubating for 30 min at room temperature with 20-nm gold-conjugated goat anti-rabbit IgG diluted 1/50 in PBS, 0.2% fish skin gelatin. Rinsed sections were then contrasted sequentially with uranyl acetate and lead citrate. Immunogold decoration was not evident when sections were incubated with preimmune serum. Images were collected using a Hitachi H-600 transmission electron microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation and characterization of a cDNA clone encoding a novel protein. The screening of an oligo(dT)-primed human placental expression cDNA library with an antibody directed against a unique peptide (see MATERIALS AND METHODS) resulted in four independent isolates, termed c4, containing identical 1.742-kb inserts. The nucleotide and deduced amino acid sequences (Fig. 1A) encoded by the 1.742-kb cDNA were found to represent a previously uncharacterized molecule based on searches of both nucleotide and protein databases. Evident in the sequence was a single, long, open reading frame of 270 amino acids, a polyadenylation signal (AATAAA), and a poly(A) tail. The lack of a consensus sequence for translation initiation (31) suggested that c4 most likely encoded the carboxy terminus of a novel protein. Searches of both the expressed sequence tag database and nonredundant nucleotide databases identified several entries matching the sequence of c4, but none provided any additional 5' sequence information.

View larger version (71K): [in this window] [in a new window]   Fig. 1.   A novel protein encoded by c4. A: partial nucleotide sequence and deduced amino acid sequence from the open reading frame are shown. Numbers refer to positions of nucleotides (top) or amino acids (bottom). A putative elastin binding sequence is underlined. Integrin recognition motif RGD sequence is shown in bold. * Stop codon position. B: secondary structure of protein was predicted using the Plotstructure function of the GCG Software Package. Hydrophilicity predictions were made according to Kyte-Doolittle, whereas turns, -helical regions, and -sheets were predicted according to Garnier-Osguthorpe-Robson. N-linked glycosylation sites (N-linked) were not present in this portion of the protein.

Both the deduced amino acid composition and primary structure of the c4 protein indicate that it is likely to be an -helical molecule (Fig. 1). The predominance of glutamic acid, alanine, and leucine residues found throughout the c4 protein (Fig. 1A) is not unusual in -helical proteins. Appropriate spacing of charged and polar side chains lends stability to -helices through the formation of salt bridges (35), whereas alanine residues often constitute the hydrophobic face of helices (4, 36). Structure prediction algorithms configured the c4 protein into two long -helical regions separated by turns (Fig. 1B). Contained within the protein is a 60 amino acid sequence with 22% homology to a microfibrillar elastin-binding peptide (underlined in Fig. 1A) (44). Also apparent is the tripeptide sequence RGD, known to mediate binding to the integrin family of cell adhesion molecules (bold letters in Fig. 1A). The carboxy terminus of the protein contains numerous prolines and is expected to form a rigid tail on the end of this predicted helical molecule. The absence of a contiguous stretch of hydrophobic amino acids suggests that the c4 protein lacks a membrane-spanning domain.

Comparison of the deduced amino acid sequence to known proteins revealed significant homologies to several structural proteins possessing -helical and coiled-coil domains. Among those with over 20% identity were the hemidesmosomal protein plectin, the bacterial import protein TolA, protein A kinase anchoring protein, and nonmuscle myosin heavy chain II. Without exception, homology with the c4 protein was limited to the glutamic acid-alanine-leucine-rich -helical domains of these proteins, implying that these primary amino acid sequences mediate folding into similar domains in functionally diverse proteins. These data are suggestive that the c4 protein exists as a rod-shaped or coiled-coil molecule.

Tissue-specific expression of the full-length c4 transcript. The distribution of the full-length c4 transcript in a panel of human tissues was determined by Northern blot analysis (Fig. 2). A c4 probe hybridized with a single transcript of ~2.5 kb in all tissues tested. The message was most abundant in placenta (Pl), pancreas (Pa), kidney (K), and skeletal muscle (M), whereas little was detected in the brain (B). Moderate expression was detected in the heart (H). These data indicate that c4 encodes a protein, which is expressed in a variety of tissue types. A probe for glyceraldehyde-3-phosphate dehydrogenase was used as a control for RNA loading and as expected a transcript of 1.26 kb was detected in all lanes but was most abundant in heart and skeletal muscle samples (Fig. 2, bottom panel).

View larger version (86K): [in this window] [in a new window]   Fig. 2.   c4 mRNA is detected in various tissues by Northern blot analysis. Top panel: a multitissue human poly(A)+ RNA blot with 2 µg RNA/lane was hybridized to a 1340-bp probe corresponding to 5' region of the c4 cDNA. H, heart; B, brain; Pl, placenta; Lu, lung; Li, liver; M, skeletal muscle; K, kidney; Pa, pancreas. Position of molecular size markers is as shown. Bottom panel: same blot reprobed with a rat GAPDH probe. Hybridization of both radioactive probes was detected after an overnight exposure to X-ray film at 80°C with an intensifying screen.

An 80,000-molecular weight protein is the product of c4 in vivo. Western blot analysis was used to analyze the molecular weight and the predicted tissue distribution of the protein encoded by c4. Polyclonal antibodies generated against purified recombinant protein encoded by c4 were used to probe an immunoblot of normal human kidney tissue extracts (Fig. 3). Both the antiserum (lane 1) and the affinity-purified antibodies (lane 3) identified a protein of 80,000 molecular weight, whereas the preimmune serum (lane 2) and immune serum preincubated with immunizing antigen (lane 4) failed to bind the immunoblot. Tissue extracts from mitral valve wall and pancreas were also found to contain this 80,000-molecular weight protein (data not shown). Thus the polyclonal antibody very specifically recognized an 80,000-molecular weight protein in tissue extracts expressing the c4 transcript. Efforts were next focused on immunolocalization of the antigen in situ.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   c4 encodes an 80,000-molecular weight protein. Tissue extracts were resolved by preparative gel SDS-PAGE and transferred to a nitrocellulose membrane. Individual strips containing 10 µg of total protein were cut and probed with lane 1, immune serum; lane 2, preimmune serum; lane 3, an affinity-purified antibody directed against recombinant c4 protein; or lane 4, same affinity-purified antibody preincubated with a 100-fold excess of immunizing antigen. Antibody binding was visualized using a horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate. Strips were carefully realigned before exposure to X-ray film. Relative migration of molecular weight standards is as indicated.

Localization of c4 protein to the vasculature. Immunofluorescence microscopy was utilized to examine the localization of the c4 protein in human kidney cortex, bovine mitral valve wall, and rat pancreas. An abundance of the protein was evidenced in the kidney cortex (Fig. 4, A and D), where it was specifically limited to the glomeruli and small (4-15 µm diameter) peritubular vessels (Fig. 4A, arrow; see also Fig. 5). Examination of mitral valve wall, a relatively homogeneous tissue consisting of the endothelium and vascular basement membrane, revealed staining of discrete curvilinear fibers oriented parallel to the direction of blood flow (Fig. 4, B and E). Furthermore, the immunostaining in a section of pancreatic tissue enriched in elastic and muscular vessels was also restricted to the vasculature (Fig. 4, C and F). These results demonstrated that the c4 protein was expressed in the vasculature, predominantly in small vessels.

View larger version (125K): [in this window] [in a new window]   Fig. 4.   c4 Protein is present in the vasculature. Thin sections of human kidney (A), bovine mitral valve wall (B), and rat pancreas (C) were stained with polyclonal antibody directed against recombinant c4 protein and visualized with a rhodamine-conjugated secondary antibody. D: hematoxylin-eosin stained section of kidney similar to that in A was visualized with brightfield microscopy. E and F: after immunofluorescence imaging, sections were stained with India ink (E) to visualize elastic fibrils or hematoxylin-eosin (F), focal fields were relocated, and tissue components visualized with brightfield microscopy. Arrows, peritubular capillaries; g, glomeruli; l, vascular lumen; a, arteriole. Magnifications: A, C, and D, ×400; B and E, ×630; F, ×350.

Antibodies to the c4 protein specifically stain microvascular elements. Double immunofluorescence studies were employed to definitively identify the vascular elements with which the 80,000-molecular weight protein was preferentially associated. Monoclonal antibody A028, directed against an unspecified endothelial cell surface protein, was used to visualize all vascular structures. Immunostaining of kidney tissue with this antibody in conjunction with the polyclonal antibody directed against the c4 protein revealed that c4 protein was associated with a subset of vascular elements (Fig. 5A). Both the glomerular capillaries as well as peritubular capillaries were positive for the c4 protein, and fortuitous oblique sections revealed staining of the juxtaglomerular mesangial matrix (data not shown). Interstitial tissue and renal tubules were devoid of immunoreactivity. At slightly higher magnification, overlap between the antigens in a subset of vessels was more readily apparent (Fig. 5B). Notably, a significant population of vessels was not stained with the antibodies directed against the c4 protein. These findings were corroborated in double-labeling experiments using antibodies directed against the circulating coagulatory protein von Willebrand factor as a marker for endothelia (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Immunohistochemical localization of c4 protein to renal microvasculature. Thin sections of human kidney tissue were stained with a polyclonal antibody directed against recombinant c4 protein and monoclonal antibody A028 directed against an unspecified endothelial cell surface antigen. Fluorophore-conjugated secondary antibodies were used to visualize c4 protein (rhodamine, red) and endothelial cells (fluorescein, green). Regions of fluorophore overlap appear yellow. g, glomerular capillaries. Magnifications: A, ×630; B, ×1,000.

The observation that c4 protein was associated with a limited population of blood vessels spurred an examination of the relative abundance of the protein in artery versus small periarterial vessels. Immunofluorescence microscopy of mesenteric artery in cross section revealed a modest deposition of the c4 protein in fibrillar arrays throughout the vascular wall of the artery. However, in sections where small periarterial vessels were also present, it was evident that the c4 protein was markedly enriched in these vessels (Fig. 6, A and B). Together these data suggested that expression of c4 protein was most prevalent in the microvasculature. In addition, the fibrillar arrangement of the c4 protein evident in large vessels indicated it may be a component of the extracellular matrix.

View larger version (79K): [in this window] [in a new window]   Fig. 6.   Differential expression of cablin in distinct vessel types. Thin sections of rat mesenteric artery were stained with a polyclonal antibody directed against recombinant c4 protein. Rhodamine-conjugated secondary antibody was used to visualize c4 protein in mesenteric artery and a small periarterial vessel. l, artery lumen. Magnifications: A, ×630; B, ×1,000.

c4 Protein is deposited in the basal laminae of capillaries. Immunoelectron microscopic analysis of kidney tissue was instrumental in establishing the precise spatial relationship between microvascular endothelial cells and the c4 protein. A low-magnification micrograph of a peritubular capillary in which the vessel lumen, an endothelial cell, the basal lamina, and a periendothelial cell are evident demonstrated an association of c4 protein with the capillary basal lamina (Fig. 7A, arrows). A higher magnification view of a similar section features the basal lamina encapsulating an endothelial cell and the process of a periendothelial cell (Fig. 7B). Gold particles decorated c4 protein throughout the extracellular matrix, notably among fibrillar material. To facilitate visualization of gold labeling, a detection antibody conjugated to large gold (20 nm) was used, which may account for the somewhat sparse labeling observed. The association of c4 protein with the fibrillar matrix surrounding capillaries prompted us to name the protein cablin (protein of the capillary basal lamina).

View larger version (144K): [in this window] [in a new window]   Fig. 7.   Immunoelectron microscopic localization of c4 protein to subendothelial basal lamina. Ultrathin sections of embedded rat kidney tissue were incubated with a polyclonal antibody directed against recombinant c4 protein and immunoreactivity was detected using gold-conjugated anti-rabbit IgG (arrows). A: low-magnification view (×6,000) of cross-sectioned capillary. B: high-magnification view (×25,000) of capillary basal lamina. en, Endothelial cell; er, erythrocyte; p, pericyte; bl, basal lamina.

Cellular expression and secretion of cablin. The close apposition between sites of cablin deposition and endothelial and periendothelial cells suggested that one or both of these cell types might be responsible for its biosynthesis. Cellular extracts and conditioned media of primary cells in culture were therefore tested for cablin expression by immunoblot analysis. Cablin was synthesized and secreted by all cell types examined, including vein endothelial cells, microvascular endothelial cells, and aortic smooth muscle cells (Fig. 8, A and B, arrow). (A slightly lower molecular weight band, seen in Fig. 8A, is thought to represent a degradation intermediate.) However, the extent of cablin secretion by different cell types was particularly noteworthy. Smooth muscle cells produced significant amounts of cablin (Fig. 8A), yet secretion appeared to be inefficient. Endothelial cells synthesized less cablin, but the majority of the protein was secreted (Fig. 8B). On the basis of these in vitro data it is plausible that relative to other cell types endothelial cells may preferentially deposit cablin in vascular extracellular matrices.

View larger version (64K): [in this window] [in a new window]   Fig. 8.   Cablin synthesis and secretion by cultured cells. Proteins in cellular extracts (A) or conditioned media (B) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with an affinity-purified antibody directed against recombinant c4 protein. Antibody binding was visualized using an HRP-conjugated secondary antibody and chemiluminescent substrate. Lane 1, aortic smooth muscle cells; lane 2, umbilical vein endothelial cells; lane 3, lung microvascular capillary endothelial cells. Equal amounts of cellular proteins were loaded (10 µg) in A, and a volume of conditioned medium corresponding to the cellular protein concentration was loaded in B. Relative migration of molecular weight standards is as indicated. Arrow, cablin.

Cablin is expressed at sites of angiogenesis. The deposition of cablin in microvascular basal laminae prompted us to examine its expression in capillaries undergoing angiogenesis. In a murine model of retinal neovascularization (56), hypoxia-induced stress results in proliferation and migration of endothelial cells normally present only in a discrete vascular layer at the periphery of the retina. This response leads to the formation of angiogenic tufts that invade the vitreous. Immunofluorescence microscopic analysis of control retinas revealed that cablin colocalized with an endothelial cell marker (Fig. 9B, arrowhead) in the vascular layer along the outer retina (Fig. 9A, asterisk). Weak cablin staining could be detected in the cell layer immediately above the photoreceptor cells, but this was barely above a diffuse background signal also observed with preimmune serum (Fig. 9E). Neither endothelial cells nor cablin were detected in the layer abutting the vitreous (containing the optic nerve fibers). In contrast to control retinas, those recovered following hypoxia displayed capillary tufts consisting of 5-10 endothelial cells growing into the vitreous (Fig. 9C, arrow), and endothelial cells were seen to permeate all layers of the retina. Although cablin was apparent in the tufts themselves (Fig. 9D, arrows), the predominant immunoreactive regions were immediately subjacent to the tufts. This finding raises the possibility that recruitment of endothelial cells to sites of hypoxic injury is correlated with the induction of cablin expression and its assembly into a supporting extracellular matrix.

View larger version (47K): [in this window] [in a new window]   Fig. 9.   Cablin is associated with sprouting capillaries in a model of angiogenesis. Variously stained thin sections of control (A and B) or experimental angiogenic mouse retinas (D and E) are shown. A: control retina stained with hematoxylin-eosin. B: polyclonal antibody directed against recombinant c4 protein and a monoclonal antibody (A028) directed against an unspecified endothelial cell surface antigen were used to stain a control retina. Cablin staining was detected using a fluorescein-conjugated secondary antibody (green), and endothelial cells were visualized with a rhodamine-conjugated secondary antibody (red). Regions of fluorophore overlap appear yellow. C: monoclonal antibody A028 directed against an unspecified endothelial cell surface antigen was used to stain an experimental retina. Endothelium was detected using a fluorescein-conjugated secondary antibody (green) and nuclei were visualized with DAPI (blue). Because of the thickness of the section, individual nuclei are visible only in the peripheral areas of the retina. D: cablin distribution in an experimental retina was detected by staining with a polyclonal antibody directed against recombinant c4 protein followed by fluorescein-conjugated secondary antibody (green) and nuclei were visualized with DAPI (blue). E: preimmune serum was used to stain an experimental retina. Antibody binding was detected with fluorescein-conjugated secondary antibody (green). V denotes vitreous; arrowhead denotes blood vessel; arrows denote capillary tufts; 1 denotes Muller's and optic nerve fiber layer and plexiform/ganglion layer; 2 denotes bipolar, horizontal, amacrine and Muller cell layer; 3 denotes photoreceptor cell layer; asterisk denotes vascular layer (this layer is incomplete in C-E). Magnification: ×400.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study describes the characterization of cablin, a novel protein preferentially associated with the capillary basal lamina. The protein was synthesized and secreted by cultured microvascular endothelial cells as well as vascular smooth muscle cells, hypothesized to be differentiated pericytes (37, 39, 48, 49). In addition, its synthesis was upregulated in tissue undergoing active neovascularization. Cablin was predominantly associated with the microvasculature, where it was specifically arrayed in the electron-lucent amorphous material of capillary basal laminae. These findings offer the first indication that the microvascular basal lamina is biochemically unique. Analyses of amino acid sequence composition and predicted secondary structure are consistent with cablin being an -helical, rodlike protein with domains characteristic of extracellular matrix proteins. The enrichment of cablin in vascular regions subject to high hydrostatic pressure and shear force (including the glomerular basement membrane and the juxtaglomerular mesangial matrix), in conjunction with a possible association with microfibrils, suggest that cablin reinforces the architectural integrity of the tissue.

The microvasculature consists of networks of capillaries, precapillary arterioles, and postcapillary venules highly specialized for the fundamental activities of molecular exchange and angiogenesis. Results presented here demonstrated that cablin was predominantly associated with the microvasculature and minimally with other vessels. Immunodetection of cablin was predicated on the relative abundance of capillaries within the sample. The glomerular and peritubular microvasculature within the microvessel-rich kidney cortex were the exclusive sites of cablin immunoreactivity. However, examination of sections enriched in elastic and muscular vessels revealed that these structures were in fact immunoreactive, albeit to a lesser extent than capillaries. Here, low levels of cablin were apparent in discrete fibrillar arrays interspersed between elastin fibrils. Although tissue etching has been instrumental in revealing masked antigenic sites on extracellular matrix proteins (22), this procedure did not alter the unprecedented distribution of cablin to a subset of vascular elements (data not shown).

The vasculature represents a continuum of vessel segments that are functionally and morphologically distinct on account of transitions in cellular phenotype. Therefore, vascular cells from diverse vessel populations were screened for cablin synthesis. Because of the difficulties associated with isolation and cultivation of pericytes, these cells could not be examined directly. However, on the basis of biochemical and morphological studies, several groups have hypothesized that pericytes differentiate into vascular smooth muscle cells (37, 39, 48, 49). Interestingly, when vascular smooth muscle cells were screened for cablin production, they were found to synthesize the greatest amount of protein among the cell types analyzed. However, microvascular endothelial cells secreted the most cablin. Therefore, it is likely that pericytes, analogous to vascular smooth muscle cells, contribute some cablin to the capillary basal lamina, but the microvascular endothelial cells are principally responsible for its deposition. Although the cellular composition of capillaries has long been recognized as unique, these data offer the first indication that the biochemical constitution of capillary basal laminae is also distinct.

The primary amino acid sequence of cablin contains two domains implicated in cell and extracellular matrix binding: an integrin-binding RGD motif and a putative elastin-binding sequence. The RGD tripeptide mediates the binding of extracellular matrix components to cellular integrins (29). RGD-binding integrins found in vascular tissue include 31 and 51. Notably, capillary endothelial cells are the only vascular cells that express 51 integrins beyond embryogenesis (41). Potential interactions between cablin and the 51 integrin may stabilize cell-extracellular matrix adherence while potentiating signaling cascades. These possibilities are currently being explored.

The second identifiable domain in cablin was a 60 amino acid stretch containing 22% identity with a bacterial elastin-binding peptide (44). Compellingly, cablin was detected in the electron-lucent regions of the basal lamina bearing resemblance to sites of elastin-associated microfibril deposition (7, 24), where it appeared in close apposition to fibrous structures. The possible association of cablin with microfibrils is particularly intriguing given the organization of microfibrils into a mechanically dynamic molecular scaffold on which effector molecules are assembled.

Because of the extensive cross-linking and organization within the extracellular matrix, microfibril proteins are notoriously difficult to remove from tissue. Sequential tissue extraction revealed the release of cablin in two pools (data not shown). Mild extraction conditions, including low levels of nonionic detergent, removed a portion of cablin, whereas the remainder of the protein was only extracted by harsh, reductive saline conditions. Cablin extracted under the mild conditions may represent a cellular or soluble extracellular form of the protein. The pool recovered on harsh extraction may correspond to molecules embedded in the extracellular matrix. Analogous solubilization conditions were necessary to disrupt the association between other secreted molecules (such as latent TGF- binding proteins and thrombospondin) and microfibrils (3, 21). On the basis of these morphological and biochemical analyses, the deposition or adsorption of cablin to microfibrils or other components of the extracellular matrix seems likely.

Capillaries serve a unique role in both angiogenesis and metabolite exchange. Detailed morphological studies have demonstrated that the architecture of the microvasculature is uniquely adapted for these functions. The structure and differentiation of the tissue is in turn dependent on the molecular composition and organization of the extracellular matrix. It is envisaged that as a unique component of the capillary matrix, cablin could function in either a structural or regulatory capacity. As a putative structural component of elastin-containing microfibrils, cablin may be important in the resistance of the tissue against mechanical injury. Such a scenario is supported by the localization of cablin to regions of high hydrostatic pressure, the extraction properties of cablin, and the likely association of cablin with components essential for structural integrity. However, it is also important to consider that angiogenic cell proliferation, differentiation, and migration are governed by the composition of this extracellular matrix (17, 26, 28, 32). Capillary sprouts are surrounded by discrete areas of extracellular matrix reorganization and synthesis (13). Therefore, the observation that cablin synthesis is induced during neovascularization is quite compelling. Conceivably, cablin could provide differentiation cues or scaffolding critical for endothelial migration during angiogenesis. Because angiogenesis is necessary for disease processes such as tumor progression and diabetic retinopathy, it is possible that regulation of cablin expression may influence the angiogenic potential of tissues involved in pathological processes. Elucidation of possible structural or regulatory roles of cablin, the first unique component of the capillary basal lamina, is likely to contribute to the understanding of processes unique to the microvasculature.