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E J Macarak

Publications and source records attributed to E J Macarak.

At least 55 records · Page 3Linked to original sources
PubMed1990

Hypoxic contraction of cultured pulmonary vascular smooth muscle cells.

The cellular events involved in generating the hypoxic pulmonary vasoconstriction response are not clearly understood, in part because of the multitude of factors that alter pulmonary vascular tone. The goal of the present studies was to determine if a cell culture preparation containing vascular smooth muscle (VSM) cells could be made to contract when exposed to a hypoxic atmosphere. Cultures containing only fetal bovine pulmonary artery VSM cells were assessed for contractile responses to hypoxic stimuli by two methods. In the first, tension forces generated by cells grown on a flexible growth surface (polymerized polydimethyl siloxane) were manifested as wrinkles and distortions of the surface under the cells. Wrinkling of the surface was noted to progressively increase with time as the culture medium bathing the cells was made hypoxic (PO2 approximately 25 mmHg). The changes were sometimes reversible upon return to normoxic conditions and appeared to be enhanced in cells already exhibiting evidence of some baseline tone. Repeated passage in culture did not diminish the hypoxic response. Evidence for contractile responses to hypoxia was also obtained from measurements of myosin light chain (MLC) phosphorylation. Conversion of MLC to the phosphorylated species is an early step in the activation of smooth muscle contraction. Lowering the PO2 in the culture medium to 59 mmHg caused a 45% increase in the proportion of MLC in the phosphorylated form as determined by two-dimensional gel electrophoresis. Similarly, cultures preincubated for 4 h with 32P and then exposed to normoxia or hypoxia for a 5-min experimental period showed more than twice as much of the label in MLCs of the hypoxic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Effects of biaxial deformation on pulmonary artery endothelial cells.

An apparatus has been designed to subject vascular cells grown on a compliant substrate in vitro to uniform, quantifiable levels of biaxial deformation. The system described can be controlled with respect to strain level, rate, and frequency to mimic the pulsatile force to which vascular cells are exposed in vivo under both physiologic and pathologic conditions. In the experiments presented here, bovine pulmonary artery endothelial cells were grown on a substrate of segmented polyurethane urea (Mitrathane). Cell growth and morphology on this substrate were compared with those of cells grown on standard tissue culture polystyrene with no difference noted between the two substrates. Primary cultures of pulmonary artery endothelial cells were seeded onto Mitrathane, which was then subjected to cyclic biaxial deformation-producing strains of 0.78%, 1.76%, 4.9%, or 12.5% at a frequency of 1 sec-1 and a duty cycle of 0.5 sec-1 for 7 h. Cells subjected to deformations generating strains of either 4.9% or 12.5% secreted significantly less fibronectin than nondeformed cells. Similar results were obtained in experiments using cloned pulmonary artery endothelial cells on Mitrathane subjected to the 4.9% strain; however, total protein synthesis was increased. Cell viability and DNA synthesis were not affected by cyclic biaxial deformation in these experiments.

Animals↗
PubMed1989

Modulation of endothelial fibronectin synthesis by polymorphonuclear granulocytes.

The attachment of polymorphonuclear granulocytes (PMNs) to vascular endothelial cells occurs continually in normal tissues; however, knowledge of the factors that control leukocyte margination is incomplete. In the present study, we used cell cultures of pulmonary artery endothelium to study their interaction with PMNs. Endothelial cells were seeded in Costar 24-well plates following which PMNs were inoculated onto the endothelial monolayers and incubated for 2 to 20 hours. During this period, fibronectin synthesis by endothelial cells was estimated by ELISA. In wells to which PMNs had been added, supernatant fibronectin concentration was increased at all time points during the 20 hour incubation. At 20 hours, supernatants from wells to which PMNs had been added contained approximately 2 1/2 times the control level of fibronectin. Since the amount of fibronectin, as determined by ELISA, adsorbed onto the added PMNs was negligible, these data suggest that PMNs can modulate the synthesis of fibronectin by pulmonary artery cells. Pulse labeling experiments and measurements of endothelial intracellular fibronectin also suggest this possibility. The endothelial response does not appear to be owing to nonspecific physical interaction since similarly sized polystyrene beads did not cause any change in supernatant fibronectin levels while glutaraldehyde-fixed PMNs caused only a 20-25% increase in fibronectin levels.

Adult↗
PubMed1989

A system to reproduce and quantify the biomechanical environment of the cell.

An in vitro system that permits application of a uniform biomechanical stimulus to a population of cells with great precision has been developed. The device is designed to subject living cells to reproducible and quantifiable biaxial strains from 0 to 10% at rates from quasi-static to 1 s-1 and frequencies from 0 to 5 Hz. Equations for determining the strain in the substrate upon which the cells are grown, based on easily measured parameters, are derived and validated experimentally. The mechanical properties of the substrate are determined, and it is demonstrated that cells can easily be cultured in the apparatus. By use of the system, cloned bovine pulmonary artery endothelial cell clones are subjected to 5% biaxial strains applied at a peak strain rate of 0.5 s-1 and a frequency of 1 Hz for 7 h with cell viability greater than 84% and cell detachment less than 8%. We demonstrate that cells must be attached to the substrate for them to be stretched and that cell strain and substrate strain are not equal. With the use of fluorescently labeled beads as cell surface markers to measure the actual strain produced in the cells as a result of the deformation of the substrate, cell elongation was found to be approximately 60% of the strain in the substrate. This constant appeared to be affected by both in vitro cell age and morphology.

Cell Physiological Phenomena↗
PubMed1989

Localization of collagen types in regional segments of the fetal bovine aorta.

Types I, III, and IV collagens were localized immunocytochemically in muscular and small arteries of fetal bovine tissues and in different regional segments of the aorta. The distribution of these collagens was found to be unique, not only with respect to collagen type and fiber orientation but also to the specific region of the vessel being examined. Collagen types I, III, and IV often codistribute in the intimal and medial regions of blood vessels, whereas types I and III show selective association in fibers with varied morphology in the adventitial layer. Specific segments of the aorta (proximal thoracic versus distal abdominal) were examined to determine the relative distribution and morphologic presentation of the collagens. In the ascending region of the aorta proximal to the heart, the types I, III, and IV collagen colocalized in the intimal and medial layers. Types I and III collagen predominated in the relatively small adventitial layer, whereas type IV collagen was found only in association with the media of small blood vessels and capillaries in this layer. In the descending thoracic region of the aorta distal to the arch, types I and IV collagens were distributed throughout the intimal and medial layers, whereas type III collagen localization was variable depending on the antibody used for detection. The adventitia stained predominantly for type III collagen that was distinctly organized in folded arrays of fibers. These same fibers stained less intensely with antibody to type I collagen. The abdominal aorta near the common iliacs stained in a fashion similar to that of muscular arteries of the organs and tissues examined, i.e., types I and IV collagen were found in the intimal and medial layers, whereas type III collagen localized heavily to the adventitial layer, with differential staining in the intima and media depending on the antibody used. Additional staining of the adventitial region of the abdominal aorta with type I and type IV collagen antibodies was the same as in the proximal and descending regions. Since the abdominal aorta sustains decreased levels of pulsatile distension compared with the ascending thoracic aorta, type III collagen distribution and its association with other matrix components may be important in regulating the range of distensibility of the vessel wall.

Animals↗
PubMed1988

Heterogeneity of collagens in rabbit cornea: type III collagen.

Whole neonate rabbit corneas and adult corneas containing 2-week-old scars were incubated in the presence of [14C] glycine. Radiolabeled collagen extracted from the corneas and scar tissue were analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis and fluorography to determine the types and relative quantity of collagen polypeptides present and synthesized by these tissues. In addition to other collagen types, type III was found in both neonate cornea and scar tissue from adult cornea, albeit in relatively small quantities. Type III collagen in normal cornea was associated with the residue after pepsin digestion and formic acid extraction of the tissue, and the same type of collagen was extracted from scar tissue after similar treatment. Type III collagen-specific monoclonal antibody bound to developing normal corneas and healing adult tissue sections, as determined by immunofluorescence. Antibody binding was localized to the endothelium and growing Descemet's membrane in fetal and neonate corneas, and restricted to the most posterior region of the corneal scar tissue. Although monoclonal antibody to keratan sulfate, used as a marker for stromal fibroblasts, bound to most of the scar tissue, the antibody failed to bind to the posterior scar tissue positive for type III collagen. We conclude that endothelial cells from fetal and neonate rabbit cornea and endothelium-derived fibroblasts from healing wounds of adult cornea synthesize and deposit type III collagen. Moreover, this collagen appears to be incorporated into the growing Descemet's membrane of normal corneas and narrow posterior portion of the scar tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Duplication of type IV collagen COOH-terminal repeats and species-specific expression of alpha 1(IV) and alpha 2(IV) collagen genes.

DNA sequencing of a 1.7-kilobase cloned cDNA allowed determination of the complete COOH-terminal noncollagenous region (NC1) of the human alpha 2(IV) collagen chain. This 227-residue domain is composed of two equal-sized sequential "repeats" as had been observed for the corresponding 229-residue domain of the alpha 1(IV) chain. Alignment of the alpha 2(IV) repeats with each other and with those in alpha 1(IV) suggests that the type IV NC1 regions evolved via an intra- to intergenic duplication. In addition, smaller internal units having a consensus sequence GYSSCLFLWYLAF are present multiple times in the 110-115-amino acid halves. Consonant with the predominance of Tyr, Leu, and Phe in the heptads, hydrophilicity profiles of the homologous alpha 1(IV) and alpha 2(IV) regions revealed extended hydrophobic stretches which are similar to those in the noncollagenous COOH terminus of the chicken alpha 1(X) chain (Ninomiya, Y., Gordon, M., van der Rest, M., Schmid, T., Linsenmayer, T., and Olsen, B. R. (1986) J. Biol. Chem. 261, 5041-5050). Isolation of an alpha 2(IV) clone also enabled us to investigate if the alpha 2(IV) and alpha 1(IV) collagen mRNAs were coordinately transcribed and if one or both were consistently associated with either type I (alpha 1 and alpha 2), III, or V (alpha 2) transcripts as a function of cell type. Northern blot hybridization of collagen cDNA probes to poly(A) RNA extracted from human and bovine cell cultures showed that only the alpha 1(III) and alpha 2(V) genes were expressed in all cells examined. Unexpectedly, neither type IV mRNAs were found in bovine endothelial, smooth muscle, or fibroblast cells, whereas both type IV species were present in human fibroblasts and to a much greater extent in human umbilical vein endothelial cells.

Amino Acid Sequence↗
PubMed1986

Susceptibility of endothelial cells derived from different blood vessels to common viruses.

We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery, thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However, when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alterations in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells.

Animals↗
PubMed1986

Production and characterization of a monoclonal antibody to human type III collagen.

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.

Amino Acids↗
PubMed1985

Herpes simplex virus type 1 infection of endothelium reduces collagen and fibronectin synthesis.

Protein synthesis was assessed in virus-infected and -uninfected cultures of bovine aorta endothelial cells by following the incorporation of [14C]proline into nondialyzable protein and by use of an ELISA assay. Monolayers were infected at confluency by incubating with herpes simplex virus type I at a multiplicity of infection (ratio of virus to cells) of 1.0, 1 hour prior to the introduction of the [14C]proline. Samples were taken from infected and uninfected cultures at various time points postinfection from both medium and cell layer fractions and analyzed for total 14C-labeled protein, 14C-labeled collagen, and 14C-labeled fibronectin. Medium proteins were analyzed by polyacrylamide gel electrophoresis, gel filtration, and electroimmunoblotting. No change was detected in total [14C] incorporation into nondialyzable protein in infected compared with uninfected endothelial cells; however, there was a significant decrease in the synthesis of collagen and fibronectin in infected cultures. The hydroxy[14C]proline content of fractionated medium proteins showed no significant differences between infected and uninfected cultures; therefore, the decrease in collagen synthesis cannot be explained by increased collagen degradation. These data suggest that infection of bovine endothelial cells causes a reduction in the synthesis of collagen and fibronectin.

Animals↗
PubMed1984

Collagen synthesis by cloned pulmonary artery endothelial cells.

Pulmonary artery endothelial cells were isolated from bovine fetal blood vessels and used for biosynthetic studies. At confluence, cultures were incubated in minimal essential medium (MEM) without serum containing [U-14C]proline. After 24 hours, medium was removed and labeled proteins were precipitated by the addition of ammonium sulfate and fractionated by diethylaminoethyl (DEAE)-cellulose chromatography. The elution profile showed four major peaks and one minor peak. Fractions within each peak were pooled, subjected to digestion by chymotrypsin and/or collagenase, and analyzed by polyacrylamide gel electrophoresis. Peak l contained a collagen which contained approximately 6% of the 3-hydroxyproline isomer while total hydroxyproline content was approximately 45%. This material was digested by purified bacterial collagenase and had a mobility slightly slower than that of alpha 1(III) which did not change under conditions that reduce disulfide bonds. Upon digestion with chymotrypsin under conditions where native procollagens are converted to alpha-chains, this material was digested. These properties suggest that this material is type VIII or EC (endothelial cell) collagen. Peak 2 contained substantial fibronectin while peak 3 contained primarily type III procollagen. The last major peak contained a mixture of collagenous and noncollagenous material. Upon digestion with chymotrypsin, several peptides were generated which were sensitive to bacterial collagenases. The two major chymotrypsin-resistant components had mobilities slower than that of alpha(III) and were not disulfide-bonded.

Animals↗
PubMed1983

Adhesion of endothelial cells to extracellular matrix proteins.

Human and bovine endothelial cells were used in a series of studies examining cell adhesion to extracellular matrix macromolecules. Collagen types I and III, an acetic acid extract of bovine lens capsule basement membrane, laminin, and fibronectin were demonstrated to affect, in varying degrees, cell adhesion, cell growth, and "sprout cell" formation in endothelial cell cultures. All the matrix macromolecules tested promoted endothelial cell adhesion, and the adhesion to the basement membrane extract was blocked by antibodies to fibronectin. When human endothelial cells were grown on defined matrices, the level of serum required for optimum growth was decreased, while bovine endothelium grown on defined matrices did not show sprout cell formation. These data suggest that endothelial cells are able to adhere to a wide variety of connective tissue components and that extracellular matrices significantly influence cell behavior.

Animals↗
PubMed1981

Fibronectin: its relationship to basement membranes. I. Light microscopic studies.

Fibronectin is a large molecular weight glycoprotein which has been shown to be associated with cell surfaces, extracellular fluids, and connective tissues. Its possible relationship with basement membranes remains controversial. To define this relationship, the distribution of this antigen was evaluated by light microscopic immunoperoxidase techniques in kidney, skin, skeletal muscle, gastrointestinal tract and parietal yolk sac carcinoma. In addition, antibodies against basement membrane and interstitial collagen were used as controls of the specificity of this reaction. Any possible cross-reactivity between plasma fibronectin and basement membrane was examined by immunodiffusion, immunoelectrophoresis and ELISA techniques. The results indicate that antibodies to plasma fibronectin do not co-localize with antibodies to basement membrane constituents. Furthermore, by immunodiffusion or ELISA, there was no cross-reactivity between plasma fibronectin and anti-basement membrane antibody, nor between basement membrane and anti-plasma fibronectin antibody. We conclude that fibronectin is probably not part of the basement membranes studied.

Animals↗
PubMed1981

Fibronectin: its relationship to basement membranes. II. Ultrastructural studies in rat kidney.

Fibronectin, basement membrane and type I collagen antigens have been localized in normal rat kidney by electron immunohistochemical methods. Immunoreactive fibronectin was found in the interstitial connective tissue matrix and on collagen fibers, while tubular, endothelial and smooth muscle basement membranes throughout the kidney were consistently negative. In the glomerulus immunoreactive fibronectin was abundant in the mesangial matrix. The peripheral glomerular basement membrane was occasionally reactive in a spotty, irregular manner. These findings suggest that fibronectin antigens are probably not a constituent of basement membranes. It is proposed that some fibronectin antigen may be trapped in the glomerular filter, and that normal glomerular cleansing mechanisms would transport this trapped fibronectin toward the mesangial areas where it would be eventually processed.

Animals↗
PubMed1981

Virus infection of endothelial cells.

Endothelial injury is important in the pathogenesis of thrombosis, atherosclerosis, disseminated intravascular coagulation, and vasculitis. The ability of several common human viruses to infect cultures of endothelial cells obtained from human umbilical veins or bovine thoracic aorta was demonstrated. Indicators of infection included cytopathology, viral growth curves, and antigen detection by immunofluorescence. Herpes simplex virus type 1, adenovirus type 7, measles virus, and parainfluenza virus type 3 infected both human venous and bovine aorta endothelium. Mumps virus, poliovirus type 1, and echovirus type 9 grew only in human venous cells; coxsackievirus B4 infected only bovine arterial cultures; and cytomegalovirus, influenza A/Victoria/75 (H3N2) virus, and respiratory syncytial virus failed to grow in either cell culture. During replication some viruses caused acute lytic changes; some produced chronic, less destructive alterations; and other induced no apparent cytopathology. The results suggest that viral replication within endothelium may be important in the pathogenesis of viral disease of initiation of vessel-wall injury.

Adenoviruses, Human↗
PubMed1980

Virus infection of endothelial cells increases granulocyte adherence.

Adherence of human granulocytes was measured on endothelial monolayers of human and bovine origin, grown in 35-mm Diam petri dishes and in cluster wells. Adherence to human endothelium in petri dishes using 1.0 ml of whole blood averaged 17.9+/-3.7%, and to bovine endothelium was 20.3+/-3.7%. Cluster wells required only 1/5 the endothelial cells needed for petri dishes, and 0.25 ml of whole blood yielded average adherence of 26.2+/-3.4 to human cells and 28.0+/-3.7 to bovine in the wells. The impact of infection of the endothelium by different viruses on subsequent granulocyte adherence was measured. Polio virus produced an acute lytic infection of human endothelial cells, with associated increased adherence to 185.4% of control 24 h after inoculation. Significantly increased adherence was noted at 6 h, before detectable cytopathic effect. Herpes simplex type I caused a similar rapidly lytic infection of bovine endothelium associated with increased adherence to 213.7% of control 6 h after inoculation. This augmented adherence could be demonstrated when granulocytes were suspended in physiologic saline solution, showing that antibody and complement need not be present. Trypsin treatment of infected monolayers did not prevent the augmentation, and supernate from infected monolayers increased the adherence of polymorphonuclear leukocytes to normal, uninfected monolayers. Chronic, slowly lytic infections, lasting 7 d or more, were induced with adenovirus in human endothelium and with measles virus in bovine cells. Adherence increased as virus was noted in the cell cultures on day 4, several days before cytotoxicity was seen. Thus, chronic viral infection of the endothelium appears possible, and results in increased granulocyte adherence. In naturally occurring disease, such an infection may act synergistically with adherent granulocytes to damage the endothelium, and may represent an in vitro model of vasculitis.

Animals↗
PubMed1979

Comparison of the intracellular lipids of cultured vascular endothelial cells and fibroblasts.

The lipids of human umbilical vein endothelial cells, calf aortic endothelial cells and foreskin fibroblasts have been compared. Cell cultures were established, and, upon confluency, the lipids were extracted and analyzed with respect to total lipid content, classes of lipids and total lipid fatty acid composition. The total quantity of lipid per milligram protein found in both human umbilical vein endothelium and calf aorta endothelium was similar to that found in fibroblasts grown in similar medium. Both types of endothelium contained the same major neutral lipid classes as fibroblasts, although they contained more phospholipid than did fibroblasts. The fatty acid composition of the three cells examined was influenced by cell type as well as the type of serum in the culture medium.

Animals↗