Retinal microvessel extracellular matrix: an immunofluorescent study.

The vasculature of the retina functions within a sheath of extracellular matrix (ECM). Unfortunately, little is known about the biochemical composition of this matrix. Abnormalities in the ECM of the retinal microvasculature are important in diabetic retinopathy as well as vasculopathies associated with connective tissue disorders. The ECM of unfixed frozen human retinal blood vessels was examined by indirect immunofluorescence using antibodies raised against collagen types I, II, III, IV, and V as well as the structural glycoproteins laminin and fibronectin. Antisera against collagen types I and IV as well as laminin and fibronectin stained a broad spectrum of retinal vessels, from large thick-walled vessels down to microvessels less than 10 micron in diameter. In contrast, antibodies against types III and V collagen were seen to stain primarily the walls of the larger vessels. Antibodies against type II collagen did not react with retinal vessels. Preincubation with the appropriate antigen or preimmune serum eliminated staining of the vessels by the antisera.

Interphotoreceptor matrix domains ensheath vertebrate cone photoreceptor cells.

The retinal interphotoreceptor matrix (IPM) occupies the space between the neural retina and the retinal pigmented epithelium (RPE), two neuroectoderm-derived epithelia. While the IPM appears to be a major route by which photoreceptor cells receive vital metabolic factors, relatively little is known concerning its structure and function. The studies reported here describe the presence of specialized domains of the IPM that ensheath cone, but not rod, inner and outer segments in pig, monkey, and human retinae. These cone extracellular matrix sheaths are chemically and structurally distinct from the remainder of the IPM as revealed by their specific binding of the lectin peanut agglutinin (PNA) and their structural stability during physical dissociation of the retina. Biochemical studies suggest that the PNA-binding components of the cone matrix sheaths are trypsin-sensitive glycoproteins. These structures may play a role in establishing a specialized microenvironment for cone photoreceptors, maintaining proper orientation of cone outer segments, and/or facilitating cone-RPE interactions.

Extracellular matrix modulation of endothelial cell shape and motility following injury in vitro.

We utilized fluorescence microscopy and affinity-purified antibodies to probe the form and function of cytoplasmic actin in endothelial cells (EC) recovering from injury and grown on extracellular matrices in vitro. Bovine aortic EC were seeded onto glass microscope coverslips that had been coated with either BSA, fibronectin, type I and III (interstitial) collagens, type IV (basement membrane) collagen or gelatin. After EC that had been grown on glass, glass-BSA or extracellular matrix-coated coverslips reached confluence, a 300-400 micron zone of cells was mechanically removed to stimulate EC migration and proliferation. Post-injury EC movements were monitored with time-lapse, phase-contrast videomicrography before fixation for actin localization with fluorescence microscopy using affinity-purified antibodies. We found that the number of stress fibres within EC was inversely proportional to the rate of movement; and, the rates of movement for EC grown on glass or glass-BSA were approximately eight times faster than EC grown on gelatin or type IV collagen (X velocity = 0.5 micron/min versus 0.06 micron/min). EC movements on fibronectin and interstitial collagens were similar (X velocity = 0.2 micron/min). These results suggest that extracellular matrix molecules modulate EC stress fibre expression, thereby producing alterations in the cytoskeleton and the resultant EC movements that follow injury in vitro. Moreover, the induction of stress fibres in the presence of basement membrane (type IV) collagen may explain the failure of aortic EC to migrate and repopulate wounded regions of intima during atherogenesis in vivo.

Alteration of cartilage matrix morphology with histological processing.

An interlacunar network in the extracellular matrix of femoral head articular cartilage of neonatal rats was seen by light microscopy to: (1) consist of elements, 0.5 micron thick, which occurred as individual elements, as bundles of elements, and as fused elements, (2) stain intensely with toluidine blue, methylene blue, and safranin O, and (3) connect chondrocytes by inserting on the chondrocyte capsules which were composed of morphologically and cytochemically similar material. By electron microscopy, the single elements were seen to be composed of thicker, denser staining areas of the honeycomb appearing matrix and the fused elements appeared as non-membrane bound channels containing granular material. Articular cartilage was processed using combinations of fixatives, dehydrating agents, and embedding media. Regardless of fixation, demineralization, or embedding, the network was not seen after dehydration of the cartilage with methanol, ethanol, acetone or tert-butanol but was seen after dehydration with aqueous solutions of glycol methacrylate, propylene oxide, 2-propanol or 2,2-dimethoxypropane. Network visualization following a variety of methods demonstrated that no single fixative, dehydrating agent, or embedding medium caused its formation. The presence of the network in different cartilage zones, its consistent morphology by light and electron microscopy, the uniformity of the elements in their connection with the chondrocytes, and presence in fresh-frozen sections suggest the network may be real, but rigorous evidence for its existence in vivo is still required. Since cartilage morphology was altered by histological methods, especially dehydration, common methods used in studying connective tissue matrix should be evaluated to determine their effect on matrix morphology.