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Biomedical subjects

I M Herman

Publications and source records attributed to I M Herman.

48 records · Page 3Linked to original sources
PubMed1987

Hemodynamics and the vascular endothelial cytoskeleton.

Although there is considerable evidence to suggest that hemodynamics play an important role in vascular disease processes, the exact mechanisms are unknown. With this in mind, we have designed a pulsatile perfusion apparatus which reproducibly delivers pulsatile hemodynamics upon freshly excised canine carotid arteries in vitro. Quantifiable simulations included normotension with normal or lowered flow rates (120/80 mmHg, 120 and 40 ml/min), normotension with lowered or elevated transmural pressures (40-170 mmHg), and elevated pulse pressure (120 and 80 mmHg) with normal (150 ml/min) or elevated rates of flow (300 and 270 ml/min). Arterial biomechanical stresses and cellular behaviors were characterized biochemically and morphologically under all these stimulations which continued for 2-24 h. We found that increased pulse pressure alone had little effect on the total amount of radiolabeled [4-14C]cholesterol present within the medial compartment. However, normotension when coupled with altered transmural pressure yielded a three- to fourfold increase. Combinations of increased pulse pressure and flow potentiated cholesterol uptake by a factor of 10 when compared with normotension control values. Simulations that enhanced carotid arterial cholesterol uptake also influenced the endothelial cytoskeletal array of actin. Stress fibers were not present within the carotid endothelial cells of either the sham controls or the normotension and increased pulse pressure (normal flow) simulations. Endothelial cells lining carotids exposed to elevations in flow or those present within vessels perfused as per simulation b above assembled stress fibers (x = 4 and 10 per cell, respectively) within the time course of these studies. When endothelial cells were subjected to hemodynamic conditions that potentiated maximally cholesterol transport, no diffuse or stress fiber staining could be seen, but the cortical array of actin was intact. These results suggest that those biomechanical stresses that alter endothelial permeability and intimal integrity may do so via cytoskeletal actin signaling.

Animals↗
PubMed1987

Characterization of avascular corneal wound healing fibroblasts. New insights into the myofibroblast.

The characteristics and derivation of corneal wound healing fibroblasts (myofibroblasts) were evaluated by studying the temporal changes in the cellular actin distribution of corneal fibrocytes following full thickness 3-mm diameter central corneal wounds in the rabbit. Under certain conditions these wounds heal without neovascularization, allowing for the detailed analysis of invading fibroblasts with minimal contamination by other cell types. The authors employed transmission electron microscopy to localize microfilaments, fluorescent microscopy using NBD-phallacidin, a mushroom toxin which binds specifically to f-actin and oligomeres of g-actin, to localize actin filaments, and isoelectric focusing gels to characterize actin isotypes. During the early stages of wound healing (1-7 days) there is a gradual change in the corneal fibrocytes adjacent to the wound margin characterized by the development of extensive rough endoplasmic reticulum, microtubules, a prominent Golgi apparatus, and a cortical microfilament network. This is in contrast to the normal fibrocyte, which, for the most part, lacks these structures. The development of microfilaments correlated with increased NBD-phallacidin fluorescence of fibrocytes adjacent to the wound as compared with fibrocytes farther removed from the site of injury. Fibroblasts appearing within the wound from 7 days to 2 months after injury had ultrastructural characteristics similar to those of myofibroblasts, including parallel arrays of microfilaments, stress fibers and cell-cell, cell-matrix attachments. Furthermore, these cells stained intensely with NBD-phallacidin, supporting the ultrastructural findings. At 1 month after injury, cells contained within the wound possessed predominantly nonmuscle isoactins (gamma) as seen by silver staining of isoelectric focusing gels, but little or no (smooth muscle) isoactins could be detected. Moreover, no significant differences could be detected between electrophoretic profiles obtained from wounded versus normal corneas. These morphologic and biochemical data suggest that the corneal fibrocyte may develop into a fibroblastlike cell similar to the myofibroblast, and is characterized by a marked increase in filamentous actin.

Actins↗
PubMed1985

Microvascular pericytes contain muscle and nonmuscle actins.

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.

Actins↗
PubMed1985

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.

Actins↗
PubMed1984

Capillary endothelial cell migration: loss of stress fibres in response to retina-derived growth factor.

While the migration of capillary endothelial cells is believed central to the process of new blood vessel development in vivo, the biochemical basis for endothelial motility is unknown. Herein, we demonstrate that retina-derived growth factor (RDGF), a mitogen for endothelial cells (EC), stimulates the migration of microvascular endothelium in vitro. The addition of RDGF directly to the culture medium causes an increase in the random movement (chemokinesis) of the EC as measured by the phagokinetic assay. Release of the factor as a gradient results in a stimulation of the directed migration (chemotaxis) of the microvascular EC. This increased EC migration is associated with a shift in morphology of the stimulated cells from a rounded to a more polarized shape. Concomitant with the RDGF-stimulated migration is a dramatic decrease in stress fibre staining visualized by immunofluorescence microscopy using affinity-purified antibodies to actin and myosin.

Actins↗
PubMed1983

Actin filament stress fibers in vascular endothelial cells in vivo.

Fluorescence microscopy with 7-nitrobenz-2-oxa-3-diazole phallacidin was used to survey vertebrate tissues for actin filament bundles comparable to the stress fibers of cultured cells. Such bundles were found only in vascular endothelial cells. Like the stress fibers of cultured cells, these actin filament bundles were stained in a punctate pattern by fluorescent antibodies to both alpha-actinin and myosin. The stress fibers were oriented parallel to the direction of blood flow and were prominent in endothelial cells from regions exposed to high-velocity flow, such as the left ventricle, aortic valve, and aorta. Actin bundles may help the endothelial cell to withstand hemodynamic stress.

Actins↗
PubMed1981

Electron microscopic localization of cytoplasmic myosin with ferritin-labeled antibodies.

We localized myosin in vertebrate nonmuscle cells by electron microscopy using purified antibodies coupled with ferritin. Native and formaldehyde-fixed filaments of purified platelet myosin filaments each consisting of approximately 30 myosin molecules bound an equivalent number of ferritin-antimyosin conjugates. In preparations of crude platelet actomyosin, the ferritin-antimyosin bound exclusively to similar short, 10-15 nm wide filaments. In both cases, binding of the ferritin-antimyosin to the myosin filaments was blocked by preincubation with unlabeled antimyosin. With indirect fluorescent antibody staining at the light microscope level, we found that the ferritin-antimyosin and unlabeled antimyosin stained HeLa cells identically, with the antibodies concentrated in 0.5-microns spots along stress fibers. By electron microscopy, we found that the concentration of ferritin-antimyosin in the dense regions of stress fibers was five to six times that in the intervening less dense regions, 20 times that in the cytoplasmic matrix, and 100 times that in the nucleus. These concentration differences may account for the light microscope antibody staining pattern of spread interphase cells. Some, but certainly not all, of the ferritin-antimyosin was associated with 10-15-nm filaments. In mouse intestinal epithelial cells, ferritin-antimyosin was located almost exclusively in the terminal web. In isolated brush borders exposed to 5 mM MgCl2, ferritin-antimyosin was also concentrated in the terminal web associated with 10-15-nm filaments.

Actomyosin↗
PubMed1981

Relation between cell activity and the distribution of cytoplasmic actin and myosin.

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.

Actins↗
PubMed1979

Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells.

We purified actin antibodies by affinity chromatography from the serum of rabbits immunized with glutaraldehyde-fixed chicken gizzard actin filaments and used this anti-actin to localize actin in myofibrils and fixed cultured cells at each stage of the cell cycle. By double immunodiffusion the anti-actin reacted with both smooth and skeletal muscle actin. Several blocking and absorption experiments demonstrated that the antibodies also bound specifically to actin in nonmuscle cells. The same structures stained using either the direct or the indirect fluorescent antibody technique; and, while the indirect method was more sensitive, the direct method was superior because there was no detectable nonspecific staining. As expected, anti-actin stained the I-band of myofibrils. It also stained stress fibers and membrane ruffles in HeLa cells. Some PtK-2 cells have straight stress fibers which stained with anti-actin, but in confluent cultures all PtK-2 cells have, instead, sinuous phase-dense fibers which stained with antibody. At prophase the whole cytoplasm stained uniformly with anti-actin. During metaphase and anaphase, anti-actin staining was concentrated diffusely in the mitotic spindle. In contrast, fluorescent heavy meromyosin stained discrete fine spindle fibers in these fixed cells. During cytokinesis, anti-actin stained the whole cytoplasm uniformly and was not concentrated in the cleavage furrow.

Actins↗
PubMedDate not supplied

Regulation of vascular smooth muscle cell growth by endothelial-synthesized extracellular matrices.

Previous work has demonstrated that aortic endothelial cells (EC) produce a heparin-like inhibitor of smooth muscle cell (SMC) growth when both cell types were cultured on plastic. We have now tested the influence of the extracellular matrix on this EC-SMC interaction. Specifically, we examined: 1) the role of different substrates (plastic, fibronectin, monomeric, and fibrillar collagens I and III, and EC-derived matrices) on the growth rate and population density of SMC; 2) the heparin-sensitivity of SMC on these diverse substrates; and 3) the effect of these same substrates on EC ability to secrete heparin-like and polypeptide inhibitors of SMC growth. SMC demonstrated a sixfold difference in sensitivity to heparin when grown on different substrates, with the following rank order: EGTA matrix greater than collagens = plastic = fibronectin greater than deoxycholic acid (DOC) matrix. Maximally, we found a 10-fold difference in the potency of the inhibitory activity secreted by EC grown on different substrates, with the following order: plastic = EGTA matrix greater than fibronectin greater than collagens = DOC matrix. Treatment of the conditioned mediums with heparinase and trypsin indicated that 58% to 76% of the inhibitory activity was due to heparin-like species, and 24% to 42% was due to protein(s). When EC cultured on EGTA matrix are compared to those pleated on DOC matrix, the potency of the heparin-like and peptide inhibitory activities increased 8- and 17-fold, respectively. Hypothetically, one would predict a 60-fold change in the potency of the antiproliferative effect if the contributions of substrate to EC production of inhibitors and SMC sensitivity were additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗