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Biomedical subjects

K T Weber

Publications and source records attributed to K T Weber.

At least 19 recordsLinked to original sources
PubMed1997

Myofibroblasts and local angiotensin II in rat cardiac tissue repair.

Tissue repair is a fundamental property of vascularized tissue. At sites of injury, phenotypically transformed fibroblast-like cells are responsible for fibrous tissue formation, expressed principally as type I and III fibrillar collagens. These cells are termed myofibroblasts because they contain alpha-smooth muscle actin microfilaments and are contractile. In vivo studies of injured rat cardiac tissues and in vitro cell culture studies have shown that such fibroblast-like cells contain requisite components for angiotensin peptide generation and angiotensin II receptors. Such locally generated angiotensin II acts in an autocrine paracrine manner to regulate collagen turnover and thereby tissue homeostasis in injured tissue.

Angiotensin II↗
PubMed1997

Identification of negative and positive regulatory elements in the rat alpha 1(I) collagen gene promoter.

Type I collagen is the main constituent of extracellular matrix found in various organs including the heart. Under some pathological conditions accumulation of excess type I collagen in the interstitium leads to organ dysfunction. In order to identify the regulatory elements in the rat alpha 1(I) collagen gene promoter, deletions were made in the promoter region. Various plasmid constructs were transfected into different fibroblasts using LipofectAMINE. The results indicated a negative cis-element between nucleotides -310 to -440 in the rat alpha 1(I) collagen gene promoter. Presence of this sequence significantly diminished the reporter gene activity. In addition we have observed that the sequence between -220 to -330 contained a positively acting cis-element, which is highly active in rat fibroblasts. Analysis of the nuclear factors binding to the negative element by electrophoretic mobility shift assays indicated that similar or identical factors are present in different fibroblasts as well as human HeLa cells and that these factors appear to bind to a composite sequence within -325 to -400. Competition with different oligonucleotides suggested that two distinct but contiguous sequence motifs may constitute the negative regulatory element. Our results with the rat alpha 1(I) collagen promoter confirm the presence of a negative cis-element previously described for the mouse promoter and provided additional information on the bipartite nature of this element.

3T3 Cells↗
PubMed1996

Effects of angiotensin II and aldosterone on collagen gene expression and protein turnover in cardiac fibroblasts.

Earlier studies have demonstrated angiotensin II (AngII) and aldosterone (ALDO) each augment cultured adult rat cardiac fibroblast (CFb) collagen synthesis. Whether this involves type I collagen, the major structural protein of the myocardium, and represents a transcriptional event, is uncertain. Accordingly, the influence of AngII and ALDO on transcription and synthesis of fibrillar collagen and on collagenolytic activity was examined in cultured CFb maintained in serum-deprived media. Using concentrations for AngII (10(-7) M) or ALDO (10(-9) M), shown to influence collagen turnover in these cells, we found: a) total collagen synthesis was significantly (p < 0.05) increased (5.4 +/- 0.41 and 4.8 +/- 0.37 vs. control 3.1 +/- 0.55); b) type I collagen production (6590 +/- 710 and 6150 +/- 410 vs. control 4700 +/- 490 ng/mL) in the medium were significantly (p < 0.01) increased; c) type I collagen mRNA expression was also significantly (p < 0.01) increased by AngII (2.0 fold) and ALDO (1.8 fold) compared with control; d) AngII, but not ALDO, significantly (p < 0.05) decreased collagenolytic activity (0.5 fold) compared with control. Thus, AngII and ALDO each increase CFb type I collagen synthesis at the level of transcription and protein synthesis and AngII, but not ALDO, alters collagenolytic activity. Such hormonally mediated alterations in CFb collagen turnover may contribute to the adverse accumulation of fibrillar collagen found in the myocardium in various disease states, where circulating AngII and/or ALDO are increased.

Aldosterone↗
PubMed1996

Triple helix-forming oligonucleotide corresponding to the polypyrimidine sequence in the rat alpha 1(I) collagen promoter specifically inhibits factor binding and transcription.

Type I and III fibrillar collagens are the major structural proteins of the extracellular matrix found in various organs including the myocardium. Abnormal and progressive accumulation of fibrillar type I collagen in the interstitial spaces compromises organ function and therefore, the study of transcriptional regulation of this gene and specific targeting of its expression is of major interest. Transient transfection of adult cardiac fibroblasts indicate that the polypurine-polypyrimidine sequence of alpha 1(I) collagen promoter between nucleotides - 200 and -140 represents an overall positive regulatory element. DNase I footprinting and electrophoretic mobility shift assays suggest that multiple factors bind to different elements of this promoter region. We further demonstrate that the unique polypyrimidine sequence between -172 and -138 of the promoter represents a suitable target for a single-stranded polypurine oligonucleotide (TFO) to form a triple helix DNA structure. Modified electrophoretic mobility shift assays show that this TFO specifically inhibits the protein-DNA interaction within the target region. In vitro transcription assays and transient transfection experiments demonstrate that the transcriptional activity of the promoter is inhibited by this oligonucleotide. We propose that TFOs represent a therapeutic potential to specifically influence the expression of alpha 1(I) collagen gene in various disease states where abnormal type I collagen accumulation is known to occur.

Animals↗
PubMed1996

Wound healing following myocardial infarction.

A wound-healing response that eventuates in fibrous tissue formation appears at the site of myocardial infarction (MI) in the affected ventricle. Fibrosis can likewise appear remote to the MI and cause an extensive structural remodeling of the myocardium of infarcted and noninfarcted ventricles. Substances involved in promoting healing at and remote to MI are of considerable interest and an important clinical issue, given that the healing response is subject to pharmacologic intervention. Angiotensin-converting enzyme (ACE) is expressed by wound-healing fibroblast-like cells; it likely serves to regulate local concentrations of angiotensin II and bradykinin involved in healing and matrix remodeling. Wound healing following MI and its regulation are addressed in this review.

Animals↗
PubMed1996

High affinity NAD(+)-dependent 11 beta-hydroxysteroid dehydrogenase in the human heart.

Receptor-ligand binding is an essential component of mineralocorticoid (MC) activity in target tissues. Detection of type 1 mineralocorticoid receptors (MR) in cardiac tissue is therefore suggestive that, like kidney, the heart is MC responsive. The presence of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) within MC responsive tissue is essential to prevent saturation of MR by glucocorticoids. Using both high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC), we have found that a high-affinity species of 11 beta-HSD predominates within human heart. Although two 11 beta-HSD isoforms were detected in human cardiac tissues, the activity of high-affinity (type 2) 11 beta-HSD was found to be at least twice that of low affinity (type 1) 11 beta-HSD. Human cardiac type 2 11 beta-HSD possesses characteristics identical to the high-affinity enzyme of distal renal tubules; 11 beta-dehydrogenation of corticosterone or cortisol to their 11-keto metabolites is NAD(+)-dependent and, with corticosterone as substrate, the enzyme has a nanomolar Km (15.1 nM as determined by Lineweaver-Burke analysis). Furthermore, its activity is unidirectional; corticosterone and cortisol are 11 beta-dehydrogenated to inactive 11-keto metabolites, whereas 11-oxoreductase activity (conversion of 11-dehydrocorticosterone and cortisone to corticosterone and cortisol, respectively) is absent. RT/PCR analysis, using primers complementary to the human renal type 2 11 beta-HSD sequence, demonstrated that the high-affinity species of 11 beta-HSD expressed in human heart is indeed the same enzyme as that produced in the kidney. These findings strongly suggest that, as is the case in the distal portion of the nephron, type 2 11 beta-HSD plays an important role in the human heart to promote glucocorticoid metabolism and to confer MC specificity upon MR.

Base Sequence↗
PubMed1996

Angiotensin converting enzyme and myofibroblasts during tissue repair in the rat heart.

Following left coronary artery ligation in the rat, markedly increased angiotensin converting enzyme (ACE) binding appears at the site of myocardial infarction (MI). This is also the case in fibrosed visceral pericardium that follows pericardiotomy alone (without MI). Immunohistochemical ACE labeling, using a monoclonal antibody, indicates fibroblast-like calls express ACE at each of these sites of tissue repair. It is unknown, however, whether these cells are phenotypically transformed fibroblasts containing alpha-smooth muscle actin (i.e. myofibroblasts). This study was therefore undertaken to determine whether myofibroblasts appear at the site of MI and pericardial fibrosis and their relationship to ACE expression. MI was created by left coronary artery ligation. Fibrosis of the visceral pericardium was induced by pericardiotomy alone. Hearts were studied on postoperative day 3, week 1, 2, 4 and 8. In serial sections of the same heart: immunohistochemistry (anti alpha-smooth muscle actin antibody and monoclonal ACE antibody, 9B9) was used to detect myofibroblasts and cells expressing ACE, respectively. We found that at sites of MI and pericardial fibrosis, myofibroblasts began to appear on day 3 and became abundant at week 1, 2, 4 and remained in these repairing sites for at least 8 weeks. Myofibroblasts at sites of MI and pericardial fibrosis are positively labeled by ACE antibody. Thus in these models of tissue repair involving either MI or pericardial fibrosis, myofibroblasts are associated with ACE expression. These findings suggest that myofibroblast ACE may play a role in the fibrogenic response of tissue repair in the rat myocardium by regulating local concentrations of substances involved in healing and matrix remodeling.

Animals↗
PubMed1996

Wound healing following myocardial infarction in the rat: role for bradykinin and prostaglandins.

Bradykinin and prostaglandins are established mediators of exudative and inflammatory phases of healing. Their contribution to the fibrogenic component of healing in the heart is less certain. We therefore undertook the present study in rats with acute myocardial infarction (MI) following left coronary artery ligation. Treatment with a bradykinin B2 receptor antagonist (Hoe140, 0.5 microgram/kg/min s.c.) or a cyclooxygenase inhibitor (indomethacin, 2 mg/kg p.o.), initiated 24 h after surgery, was examined for responses in MI topography (size and area), MI and nonMI tissue fibrosis (fibrillar collagen specific picrosirius red). Early (week 1) and late (week 4) phases of fibrogenesis postMI were examined. Compared to control, we found: (1) MI size at weeks 1 and 4 was comparable in untreated and treated rats: (2) infarct area, a measure of scar thickness, was reduced (P < 0.05) at week 4 by each intervention; and (3) densitometric collagen volume fraction did not reveal a reduction in collagen accumulation at the MI site, but this was evident remote to the MI (P < 0.05) at week 4 for each agent. Thus, pharmacological interference with bradykinin-receptor binding or prostaglandin synthesis following MI is associated with reduced fibrillar collagen formation. Though the mechanism responsible for observed alteration in fibrogenesis is uncertain, anti-inflammatory and anti-proliferative properties of these agents may be responsible.

Analysis of Variance↗
PubMed1996

In situ production of angiotensin II by fibrosed rat pericardium.

Following pericardiotomy in rats, subsequent fibrosis of the visceral pericardium becomes a site of high-density angiotensin-converting enzyme (ACE) binding. This study was undertaken to determine whether this exteriorized site of ACE activity is associated with angiotensin II (AngII) production. Four weeks after pericardiotomy, hearts were isolated and maintained by Krebs-Henseleit perfusion: coronary venous and Thebesian drainage were removed by cannulae. Following a 30-min period of stabilization, a balloon containing superfusate was placed around the heart. Superfusate composition was controlled and included either lisinopril (10(-7) mol/l), angiotensin I (AngI, 10(-7) mol/l), or angiotensinogen (10(-6) mol/l). Sixty min later, superfusate AngII concentration was determined (high-performance liquid chromatography followed by radioimmunoassay). Pericardial fibrosis was confirmed by picrosirius red staining and its high-density ACE binding by quantitative in vitro autoradiography (125I-351A). ACE activity was measured by hippuryl-histidyl-leucine degradation. In coronary effluent, AngII concentration and ACE activity were not different between controls and hearts with pericardial fibrosis. Compared to unoperated, age/sex-matched control hearts, however, we found those with pericardial fibrosis to have: (a) significantly (P < 0.05) greater tissue ACE activity (118.42 +/- 6.66 v 89.45 +/- 7.70 nmol/min/g): (b) significantly (P < 0.01) greater superfusate AngII concentration (4.98 +/- 0.94 v 1.43 +/- 0.28 pg/ml); (c) lisinopril markedly attenuated superfusate AngII concentration to that seen in controls; (d) exogenous AngI markedly increased AngII production (13.76 +/- 1.65 v 4.98 +/- 0.94 pg/ml); and (e) exogenous angiotensinogen did not alter superfusate AngII. Thus, high-density ACE binding and ACE activity of fibrosed pericardium is responsible for AngII production in this in vitro model. Cells involved in generating AngI at this site are uncertain and may involve fibroblast-like cells that express ACE and have ACE activity. The role of local AngII production is unknown, but its autocrine/paracrine properties may regulate collagen turnover of these cells.

Angiotensin I↗
PubMed1996

Valvular interstitial cells express angiotensinogen and cathepsin D, and generate angiotensin peptides.

Cells capable of de novo angiotensin (Ang)II generation in the heart remain unidentified. High-density angiotensin converting enzyme (ACE) binding has been localized to sites of high collagen turnover, such as heart valve leaflets and their valvular interstitial cells (VIC). VIC express ACE mRNA and their membrane-bound ACE utilizes AngI as substrate. Whether VIC also express angiotensinogen (Ao) and an aspartyl protease, and whether they generate AngI and II de novo, is presently unknown. We sought to address these questions in serum-deprived cultured VIC. Ao, renin and cathepsin D (Cat-D) mRNA expression was addressed by RT-PCR. Production of Ao, AngI and AngII peptides were measured in VIC-culture media by radioimmunoassay (RIA). Immunoreactive Cat-D was detected by immunofluorescein labeling and Western blotting. Cat-D and renin activities were determined by spectrofluorometric and autoradiographic methods and AngI generation by RIA. Results showed (a) expression of Ao and Cat-D both at mRNA and protein levels; (b) AngI and AngII peptides in culture media; (c) acceleration of AngII production by exogenous AngI (1 nmol/l), which was blocked by lisinopril (0.1 mumol/l); (d) that dexamethasone (0.1 mumol/l) increased AngII production; (e) a 46 kDa immunoreactive Cat-D protein by Western blotting; (f) aspartyl protease activity, using chromogenic and 125I-labeled Ao as substrates, inhibited by pepstatin-A; and (g) the absence of renin mRNA and activity. It is concluded that at both the mRNA and protein levels, cultured VIC express Ao and Cat-D, and can generate AngI and AngII peptides by the action of a non-renin protease Cat-D and ACE, respectively. VIC therefore appear to represent a constitutive nonendothelial cell found in adult rat heart valve leaflets, which are capable of de novo Ang peptide generation.

Angiotensin I↗
PubMed1996

Angiotensin-converting enzyme and wound healing in diverse tissues of the rat.

Autoradiographic binding density of angiotensin-converting enzyme (ACE), an indirect measure of ACE activity, is markedly increased at sites of fibrous tissue that appear in the injured heart. This includes myocardial infarction (MI) caused by left coronary artery ligation; endocardial fibrosis of the interventricular septum and perivascular fibrosis of intramyocardial coronary arterioles of the right ventricle, each of which appear remote to MI; and pericardial fibrosis after pericardiotomy (without MI). Expressed in fibroblast-like cells found at each site of tissue repair, ACE may be common to tissue repair in the rat heart, irrespective of the etiologic basis of injury. To address this hypothesis and to determine whether this also applies to other tissues (skin and kidney), the present study was undertaken. ACE binding density was measured by quantitative in vitro autoradiography (125I-351A) in injured rat heart, skin, and kidney. Experimental observations included foreign-body fibrosis after placement of silk ligature in skin or myocardium, endomyocardial myocyte necrosis and fibrosis that accompanied isoproterenol administration (1 mg/kg sc x 2 days), and embolic infarction of the kidney as a result of mural thrombus of the left ventricle that appeared after anterior MI. Fibrosis was identified by collagen-specific staining with picrosirius red. Hematoxylin-eosin staining and immunohistochemical labeling with alpha-smooth muscle actin (alpha-SMA) antibody were used to address cell morphology and phenotype, respectively. We found (1) endomyocardial fibrosis 2 weeks after isoproterenol; (2) fibrosis surrounding silk suture in heart and skin 1 week after placement; (3) renal infarction 1 week after left coronary artery ligation; (4) numerous fibroblast-like cells containing alpha-SMA, as well as macrophages, at sites of repair in all tissues studied; and (5) markedly increased ACE binding density at each of these sites. Thus ACE is integral to tissue repair in the heart, skin, and kidney of the rat, irrespective of the etiologic basis of injury. At these sites ACE may serve to regulate local concentrations of substances involved in tissue repair.

Animals↗