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Biomedical subjects

M Rabinovitch

Publications and source records attributed to M Rabinovitch.

At least 55 records · Page 3Linked to original sources
PubMed1998

Elastase and the pathobiology of unexplained pulmonary hypertension.

Our laboratory has focused on the increased activity of an endogenous vascular elastase in the pathobiology of pulmonary hypertension and on the mechanisms by which it is upregulated and by which it orchestrates abnormal remodeling of the vessel wall, specifically the induction of growth factors, the induction of the glycoprotein tenascin, which amplifies the proliferative response, and fibronectin, which is critical to the process of smooth muscle migration in the context of neointimal formation. We explore strategies by which targetting these processes might arrest progression or induce regression of pulmonary vascular disease associated with unexplained pulmonary hypertension.

Animals↗
PubMed1998

alpha1-Proteinase inhibitor therapy for the prevention of chronic lung disease of prematurity: a randomized, controlled trial.

BACKGROUND: An imbalance between increased neutrophil elastase and a decreased antiprotease shield has been suggested as a factor contributing to the development of chronic lung disease (CLD). We hypothesized that administration of alpha1-proteinase inhibitor (A1PI), also known as alpha1-antitrypsin, to premature neonates would prevent CLD. DESIGN: A randomized, placebo-controlled, prospective study of A1PI supplementation was performed. Neonates <24 hours of age with birth weights 600-1000 g on respiratory support, and 1001-1250 g with respiratory distress syndrome (RDS) were eligible. Intravenous A1PI (60 mg/kg) or placebo was infused on days 0, 4, 7, and 14. Primary outcome was CLD in survivors, defined as the need for supplemental oxygen on day 28. RESULTS: A total of 106 patients were recruited. There were no significant differences between groups in birth weight or incidence of RDS. The incidence of CLD in survivors was lower in the treated group, but the difference did not reach statistical significance (relative risk [RR], 0.79; confidence interval [CI], 0.60-1.02). This beneficial trend persisted at 36 weeks corrected gestational age (RR, 0.48; CI, 0.23-1.00). The incidence of pulmonary hemorrhage was lower in the treated group (RR, 0.22; CI, 0.05-0.98). Other complications were not significantly different between groups. CONCLUSIONS: In this, the first trial of a protease inhibitor for the prevention of CLD in premature infants, the infusions were well-tolerated. A1PI therapy may impede the development of CLD and appears to reduce the incidence of pulmonary hemorrhage in some neonates born prematurely.

Birth Weight↗
PubMed1997

Microtubule-associated protein 1 light chain 3 is a fibronectin mRNA-binding protein linked to mRNA translation in lamb vascular smooth muscle cells.

Intimal cushions form in the fetal ductus arteriosus by fibronectin-dependent smooth muscle cell migration which is associated with greater efficiency of fibronectin mRNA translation. We investigated whether the AU-rich element (ARE), UUAUUUAU, in the 3'-untranslated region (3'UTR) of fibronectin mRNA is involved in this mechanism by transfecting smooth muscle cells with plasmids containing the chloramphenicol acetyltransferase coding region with its 3'UTR replaced by fibronectin 3'UTR bearing intact or mutated ARE. More efficient translation of fusion mRNA with intact versus mutated ARE was observed. This effect was amplified in ductus (10.9-fold) compared with nonmigratory, lower fibronectin-producing aorta cells (6.5-fold). Ductus cells transfected with wild-type but not ARE-mutated plasmid reverted to the stellate phenotype of aorta cells associated with reduced fibronectin production. This suggested that plasmid ARE sequesters RNA-binding factors, thereby reducing endogenous fibronectin mRNA translation. We next purified a 15-kD fibronectin ARE-dependent RNA-binding protein and identified it as microtubule-associated protein 1 light chain 3 (LC3). LC3 is present in greater amounts in ductus compared with aorta cells, and overexpression of LC3 in aortic cells by transfection enhances fibronectin mRNA translation to levels observed in ductus cells.

Amino Acid Sequence↗
PubMed1997

Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 integrin to promote epidermal growth factor receptor phosphorylation and growth.

Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matrix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a beta 3 integrin- dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and "rescues" cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with alpha v beta 3 integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC beta 3 integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating beta 3 integrin ligands in type I collagen. In turn, alpha v beta 3 interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.

Actins↗
PubMed1997

Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone.

1. We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450-based mono-oxygenase reaction controlling the formation of endothelin-1 (ET-1). In the present study, we have examined whether CO is formed naturally in the vessel. 2. The CO-forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO-2) and inducible (HO-1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO-1 was localized to endothelial and muscle cells, while HO-2 was found only in muscle cells. Inside the muscle cells, HO-1 and HO-2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO-1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3. CO was formed enzymatically from added substrate (hemin, 50 microM) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 microM). 4. ZnPP (10 microM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin-treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2). 5. ZnPP (10 microM) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 microM)-contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6. We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.

Age Factors↗
PubMed1997

Tenascin-C, proliferation and subendothelial fibronectin in progressive pulmonary vascular disease.

Progressive pulmonary hypertension is characterized by smooth muscle cell proliferation and migration leading to occlusive arterial lesions. Previously, using cultured smooth muscle cells, we demonstrated that epidermal growth factor (EGF)-dependent proliferation and migration are dependent on tenascin-C (Tn) and cellular fibronectin (Fn), respectively. In this study we applied immunohistochemistry to lung biopsy tissue from patients with congenital heart defects and pulmonary hypertension to determine how the distribution and intensity of Tn, EGF, proliferating cell nuclear antigen (PCNA), and Fn expression related to arterial abnormalities. With mildly increased wall thickness, minimal Tn, PCNA, and EGF was evident. With progressive hypertrophy, moderately intense foci of Tn were apparent in the adventitia, periendothelium, and occasionally the media but not consistently co-distributing with EGF and PCNA. With obstructive lesions, intense neointimal Tn expression co-localized with EGF and PCNA. Fn accumulation in the periendothelium increased with medial hypertrophy and became more widespread in a diffuse pattern with neointimal formation. The neointima was predominantly composed of alpha-smooth-muscle-actin-positive cells, occasional inflammatory cells with no evidence of apoptosis. These studies are consistent with Tn modulating EGF-dependent neointimal smooth muscle cell proliferation and Fn providing a gradient for smooth muscle cell migration from media to neointima.

Adolescent↗
PubMed1996

Elafin, a serine elastase inhibitor, attenuates post-cardiac transplant coronary arteriopathy and reduces myocardial necrosis in rabbits afer heterotopic cardiac transplantation.

We have related experimentally induced post-cardiac transplant coronary arteriopathy to increased elastolytic activity, IL-1beta, fibronectin-mediated inflammatory and smooth muscle cell (SMC) migration, and SMC proliferation. Since our in vitro studies show that a serine elastase releases SMC mitogens and facilitates IL-lbeta induction of fibronectin, we hypothesized that administration in vivo of the specific serine elastase inhibitor, elafin, would decrease the post-cardiac transplant coronary arteriopathy. Cholesterol-fed rabbits underwent a heterotopic cardiac transplant without immunosuppression and received elafin (1.79 mg/kg per d continuous infusion after a 9 mg bolus, n = 6) or vehicle (n = 6). 1 wk later, hearts were harvested for morphometric, immunohistochemical, and biochemical analyses. A > 70% decrease in the total number of coronary arteries with intimal thickening in elafin-treated compared to control donor hearts (P < 0.002) was associated with reduced vascular elastolytic activity judged by fewer breaks in the internal elastic lamina (P < 0.03), less accumulation of immunoreactive fibronectin (P < 0.02), and reduced cell proliferation quantified by proliferating cell nuclear antigen (P < 0.0001). Despite myocardial lymphocytic infiltration, wet weight of elafin-treated donor hearts was reduced by 50% compared to untreated controls (P < 0.002) and associated with relative preservation of myocyte integrity, instead of extensive myocardial necrosis (P < 0.004). This protective effect correlated with decreased myocardial elastolytic activity (P < 0.0001) and inflammatory cell proliferation (P < 0.0001) and with an elafin-inhibitable elastase in lymphocytes. Serine elastase activity thus appears an important therapeutic target for post-cardiac transplant coronary arteriopathy and myocardial necrosis induced by rejection.

Animals↗
PubMed1996

Functional interplay between interleukin-1 receptor and elastin binding protein regulates fibronectin production in coronary artery smooth muscle cells.

We have previously shown that free galactosugars and N-acetylgalactosamine glycosaminoglycans, e.g., chondroitin sulfate (CS), release the 67-kDa elastin binding protein (EBP) from arterial smooth muscle cell (SMC) surfaces. This disrupts cell contact with elastin, impairs assembly of new elastic fibers, and increases fibronectin production, all of which promote SMC migration and intimal thickening. The present study uncovered a mechanism regulating fibronectin production in vascular myocytes related to a functional interplay between EBP and the interleukin-1 receptor type I. We showed that CS-induced shedding of the EBP or internalization of this receptor after saturation with elastin-derived peptides (kappa-elastin, kappa-El) stimulated fibronectin production in cultures of coronary artery SMC to a level observed with recombinant interleukin (IL)-1beta. Upregulation of fibronectin by CS or kappa-El was abolished by a soluble IL-1 receptor antagonist, and synergistic stimulation of fibronectin production occurred when CS or kappa-El was added with IL-1beta. Immunohistochemistry showed that EBP and IL-1 receptor type I codistributed on surfaces of unstimulated coronary artery SMC, while CS- and kappa-El-dependent removal of EBP from the cell surface increased binding of radiolabeled IL-1beta to CA SMC. We propose a unique interaction between both receptors in which unoccupied EBP interferes with IL-1beta binding. Conversely, increased accumulation of N-acetylgalactosamine glycosaminoglycans or elastin-derived peptides in the vascular wall may unmask IL-1 receptor type I and increase binding of the cytokine and consequent upregulation of fibronectin production.

Acetylgalactosamine↗
PubMed1996

Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular-matrix bound basic fibroblast growth factor.

There is increasing evidence that extracellular matrix (ECM)-degrading proteinases contribute to the process of medial hypertrophy and neointimal proliferation in pulmonary vascular diseases. However, little is known about how proteinases, specifically elastases, induce vascular smooth muscle cell (SMC) hyperplasia. Our objective was to determine whether exogenous human leukocyte elastase (HLE), as well as endogenous vascular elastase, could release basic fibroblast growth factor (bFGF), a potent mitogen stored in the ECM surrounding SMCs. Cultured ovine and porcine pulmonary artery SMC were pre-incubated with [125I]-bFGF. After removal of unbound [125I]-bFGF, administration of HLE (0-1.0 microgram /ml, 1 h) resulted in a concentration-dependent accumulation of [125I]-bFGF in the conditioned medium, mirrored by depletion from the ECM. The serine elastase inhibitor elafin blocked this HLE-mediated action. Assessment by Western immunoblotting further demonstrated that HLE evoked the release of ECM-bound endogenous bFGF. When incubated with serum-starved SMC, conditioned medium from HLE-treated cells stimulated [3H]-thymidine incorporation, a feature neutralized by bFGF antibodies. In addition, SMC exposed to serum treated elastin (STE), previously shown to stimulate endogenous vascular elastase, liberated bioavailable bFGF from ECM stores, as determined by autoradiography, Western immunoblotting, and stimulation of DNA synthesis and SMC proliferation. Chondroitin sulfate, an inhibitor of STE-induced elastase activity, attenuated the release of bFGF. Our studies demonstrate that HLE, secreted by inflammatory cells, and endogenous vascular elastase release matrix-bound bFGF, suggesting a mechanism whereby elastases, through degradation of ECM, induce SMC proliferation associated with progressive vascular disease.

Animals↗
PubMed1996

Cohabitation of Leishmania amazonensis and Coxiella burnetii.

Intracellular pathogens customize the composition and function of the vacuoles they occupy, and can arrest or distort vacuolar maturation. In doubly infected cells, vacuoles that contain two different parasites can be used to test for exclusionary mechanisms, for expression of vacuolar phenotypes that permit or restrict fusion, and for the survival of pathogens targeted to an unusual cellular compartment.

Animals↗
PubMed1996

Cell-extracellular matrix interactions in the ductus arteriosus and perinatal pulmonary circulation.

Our studies and those of others have shown that changes in the extracellular matrix have profound effects on vascular remodeling. In the ductus, increased production of endothelial hyaluronan and smooth muscle cell chondroitin sulfate and fibronectin and impaired elastin fiber assembly are features critical to smooth muscle cell migration into the subendothelium and intimal cushion formation. There is a developmentally orchestrated process that involves post-transcriptional mechanisms of gene regulation. Closure of the ductus arteriosus is associated with further changes in matrix expression and programmed cell death. The changes in the extracellular matrix that induce neointimal formation are also observed in pathological conditions in pulmonary and coronary arteries. Plasticity of the pulmonary circulation in the perinatal period also involves matrix regulation, and processes that prevent the normal decrease in pulmonary vascular resistance will result in impaired matrix regulation, and in the development of structural changes in the pulmonary arteries, including abnormal smooth muscle cell differentiation, hypertrophy and proliferation, which sustain the elevation in pulmonary artery pressure.

Animals↗
PubMed1996

Tenascin-C is induced with progressive pulmonary vascular disease in rats and is functionally related to increased smooth muscle cell proliferation.

Tenascin-C, an extracellular matrix glycoprotein prominent during tissue remodeling, has been linked to cell migration, proliferation, and apoptosis. To determine its potential role in the pathobiology of pulmonary hypertension, we compared tenascin expression in adult and infant rat pulmonary arteries (PAs) after injection of the toxin monocrotaline. Immunohistochemistry, in situ hybridization, and Northern blot analysis demonstrated induction of tenascin in adult rat central and peripheral PA. Tenascin was not, however, detected in infant vessels, which show spontaneous regression of vascular lesions. To determine a function for tenascin, we correlated its expression with evidence of apoptosis and cell proliferation using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and 5-bromo-2'-deoxyuridine labeling, respectively. Apoptosis was observed only in the adult rat PA endothelial cell layer, preceding the induction of tenascin, which colocalized both temporally and spatially with proliferating smooth muscle cells (SMCs). A cause-and-effect relationship was documented in cultured rat PA SMCs, where tenascin promoted growth in response to basic fibroblast growth factor and was a prerequisite for epidermal growth factor-induced proliferation. These data provide novel functional information suggesting that endothelial cell apoptosis precedes progressive pulmonary hypertension and that induction of tenascin may be critical to growth factor-dependent SMC proliferation.

Animals↗
PubMed1996

Fusion of Leishmania amazonensis parasitophorous vacuoles with phagosomes containing zymosan particles: cinemicrographic and ultrastructural observations.

Studies on fixed preparations have shown that vacuoles containing zymosan (Z) particles internalized by infected macrophages can selectively fuse with the large parasitophorous vacuoles (PVs) that shelter Leishmania amazonensis. To examine the kinetics of vacuolar fusion in individual cells, particles were followed by time-lapse cinemicrography from their uptake to their entry in a PV. Newly formed Z-containing vacuoles moved centripetally and, if they contacted a PV, the two vacuoles remained closely apposed for variable, often extended, periods of time before they eventually fused. Transmission electron microscopy confirmed that the cytoplasm separating the partner vacuoles could be reduced to a very thin layer. Initiation of fusion was indicated by reduced refractility of the boundary between Z vacuoles and target PVs. Within a few minutes the PV enlarged and encompassed the Z particles, which remained immobile throughout. The interval between phagocytosis and fusion, 50 +/- 7.4 min (N = 17; range, 4 to 108 min), suggests that most but not all Z vacuoles underwent significant maturation by the time of fusion. Some particles were transferred singly, others entered PVs in groups of 2 or more, and additional clustered transfers to the same vacuole were also observed. These observations provide a baseline for studies of the biochemical mechanisms and the pharmacological control of the fusion of Leishmania PVs, and for the comparison of the fusion behavior of the PVs with that of other phagocytically derived vacuoles.

Animals↗
PubMed1995

Reciprocal induction of tumor necrosis factor-alpha and interleukin-1 beta activity mediates fibronectin synthesis in coronary artery smooth muscle cells.

We previously demonstrated an immune-inflammatory response associated with increased expression of interleukin (IL)-1 beta and fibronectin in graft coronary arteriopathy in piglets following heterotopic heart transplant. Further studies showed that increased endogenously produced IL-1 beta was upregulating fibronectin production by donor coronary artery (CA) smooth muscle cells (SMC). Since co-induction of IL-1 beta and tumor necrosis factor (TNF)-alpha has been shown in other systems, we investigated the possible interaction between these cytokines in regulating fibronectin production in CA SMC. First, we documented increased TNF-alpha expression in vivo in donor compared to host CA. Next, synthesis of fibronectin was measured in host and donor CA SMC following [35S]-methionine radiolabeling and gelatin-sepharose extraction. As previously shown with IL-1 beta, increased donor CA SMC fibronectin synthesis was reduced to host levels in the presence of TNF-alpha antibodies, and exogenous TNF-alpha upregulated fibronectin synthesis in host CA SMC to levels in donor cells. In normal CA SMC, TNF-alpha-stimulated fibronectin production was downregulated to or below control levels in the presence of IL-1 beta antibodies. Likewise, IL-1 beta-stimulated fibronectin synthesis was downregulated to control levels when TNF-alpha neutralizing antibodies were added. Combining TNF-alpha and IL-1 beta enhanced fibronectin production over that observed with either cytokine alone, but was not additive. Thus, our studies suggest that vascular SMC fibronectin synthesis is regulated by reciprocal induction of IL-1 beta and TNF-alpha activity and provide the first demonstration of a 'cytokine loop' modulating matrix production.

Animals↗