INCREASED EXPRESSION OF BIGLYCAN mRNA IN PRESSURE-OVERLOADED RAT HEART

Biglycan mRNA expression in rat myocardium after abdominal aortic banding with renal ischemia was examined. The Northern blot analysis demonstrated that expression of biglycan mRNA in the pressure-overloaded hearts on days 2, 7, 14 and 28 was 2.88 ± 0.89, 2.32 ± 0.49, 2.17 ± 0.57 and 1.81 ± 0.46-fold higher, respectively, than that in the sham-operated hearts. In situ hybridization showed an increased density of biglycan mRNA signal-positive cells in the pressure-overloaded hearts. The cells with positive signals were spindle-shaped mesenchymal cells in the myocardial interstitium. A marked increase in biglycan mRNA signal expression was also observed in endothelial cells and smooth muscle cells of the thickened myocardial capillary wall. These results demonstrated an increase in biglycan mRNA in the pressure-overloaded heart in mesenchymal cells in the myocardial interstitium, and in endothelial and smooth muscle cells of the capillaries, indicating that biglycan contributes to the ventricular and vascular remodeling in response to pressure overload.     

Embryonic smooth muscle myosin heavy chain SMemb is expressed in pressure-overloaded cardiac fibroblasts

Left ventricular hypertrophy (LVH) is a secondary adaptation to increased external load. Various qualitative and quantitative changes in myocytes and extracellular components occur during the development of LVH. It has recently been demonstrated that alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts appear in the interstitium of the heart subjected to increased workload suggesting that cardiac fibroblasts as well as myocytes alter their phenotype in response to pressure overload. In the present study, to explore the load-induced response and phenotypic modulation of cardiac fibroblasts, the localization of embryonic smooth muscle myosin heavy chain (SMemb) and alpha-SMA in thoracic aorta-constricted rat hearts was investigated by immunohistochemistry, and the morphology of the SMemb-expressing cells was examined by electron microscopy. In addition, to clarify the mechanisms by which SMemb is induced in pressure-overloaded hearts, mRNA expression of SMemb in aorta-constricted rat hearts and in transforming growth factor-beta1 (TGF-beta1)-treated or mechanically-stretched cultured cardiac fibroblasts was investigated. Enhanced staining of SMemb and alpha-SMA was detected in the interstitial spindle-shaped cells in the fibrotic lesions of the pressure-overloaded left ventricles by immunohistochemistry. These cells were demonstrated by electron microscopy to have features specific for activated fibroblasts such as serrated nuclei or prominent rough endoplasmic reticulum. These cells also had characteristic features of myofibroblasts, i.e. irregularly arranged actin filaments and scattered dense bodies. Northern blot analysis revealed increased mRNA levels of SMemb both in aorta-constricted rat hearts and in cultured cardiac fibroblasts stimulated by TGF-beta1 or by mechanical stretch. These results suggest that SMemb may be a molecular marker both for the detection of activated cardiac fibroblasts that may play important roles in the remodeling of pressure-overloaded cardiac interstitium, and for the identification of the regu latory mechanisms that control the phenotypic modulation of cardiac fibroblasts in response to pressure overload.     

Expression of vascular endothelial growth factor in human myocardial infarction

Summary To define mechanisms that may influence collateral circulation and angiogenesis, we investigated vascular endothelial growth factor (VEGF) mRNA expression in human hearts. In non-ischemic human hearts, VEGF mRNA was not detected in vessels, but was found in cardiomyocytes. In hearts with myocardial infarction, the intensity of the VEGF signal was much higher in smooth muscle cells of arterioles adjacent to necrosis and in infiltrating macrophages than in myocytes around the site of the necrosis. This study suggests that levels of VEGF expression are high in smooth muscle cells and macrophages around infarcted areas after myocardial infarction and that VEGF may play a role in promoting collateral circulation and angiogenesis in human ischemic hearts.     

Induction of myocardial biglycan in heart failure in rats--an extracellular matrix component targeted by AT(1) receptor antagonism

OBJECTIVE: Cardiac remodelling associated with congestive heart failure typically involves dilatation of the ventricular cavities, cardiomyocyte hypertrophy and alterations of extracellular matrix. Biglycan is an extracellular proteoglycan with several recently appreciated functions including cell adhesion, collagen fibril assembly, and growth factor interactions. The aims of this study were to investigate the regulation of biglycan expression and to elucidate the site(s) of synthesis of biglycan in myocardial tissue in an experimental model of heart failure (HF). METHODS: Myocardial tissue samples were obtained from rats with myocardial infarction (MI) subsequent to ligation of the left coronary artery. Northern blot analysis and real-time quantitative RT-PCR were employed to investigate mRNA levels. The cellular distribution of biglycan was analysed by in situ hybridisation and immunohistochemistry. RESULTS: Myocardial biglycan mRNA levels in non-ischemic tissue of both left and right ventricles of heart failure rats were substantially elevated as compared to sham-operated rats. Although expression levels peaked 7 days after MI (13-fold increase compared to the sham group, P<0.05), substantial elevations of biglycan mRNA were observed throughout the study period. Analysis of cellular distribution revealed that biglycan expression was confined to myocardial fibroblasts and vascular endothelial cells. In cardiac fibroblasts isolated from failing hearts, biglycan mRNA levels were markedly elevated compared with fibroblasts from sham-operated rats. In addition, in rats with ischemic heart failure treatment with the AT(1) receptor antagonist losartan (12.5 mg.kg(-1) b.i.d. per os, for 25 days) prevented the increase of myocardial biglycan as well as TGF-beta(1) mRNA. CONCLUSION: This report demonstrates global induction of myocardial biglycan mRNA in heart failure. Myocardial biglycan expression could be targeted by AT(1) receptor antagonism, an intervention well documented to halt cardiac remodelling in heart failure. Furthermore, the study provides evidence that angiotensin II is a regulator of biglycan expression in cardiac fibroblasts.     

Extracellular matrix glycoprotein biglycan enhances vascular smooth muscle cell proliferation and migration

Proteoglycans are produced and secreted by vascular smooth muscle cells, but the pathophysiological role of these glycoproteins in the vasculature is an enigma. Because the small leucine-rich proteoglycan (SLRP) biglycan is overexpressed in arteriosclerotic lesions, we produced mice constitutively overexpressing biglycan in the vascular smooth muscle, in order to examine the effects on vascular pathology. In the aorta and renal vasculature, increased vascular proliferation was seen both in the basal state and after infusion of angiotensin II (Ang II) in the transgenic mice compared with wild-type controls. In addition, the combination of biglycan overexpression and Ang II infusion resulted in marked increases in vascular smooth muscle cell proliferation and migration in the coronary arteries, as well as increases in fibrosis surrounding the vessels. In vitro, biglycan caused an increase in thymidine incorporation and migration of vascular smooth muscle cells, whereas these parameters were unchanged or reduced in endothelial cells. Moreover, addition of biglycan resulted in an increase in cdk2 expression and decrease in p27 levels in the vascular smooth muscle cells. These results suggest that this extracellular matrix SLRP may be involved in the regulation of vascular smooth muscle growth and migration through cdk2- and p27-dependent pathways. Furthermore, changes in biglycan expression could be a factor influencing the susceptibility of arteries to vascular injury, and may play a direct role in the pathogenesis of vascular lesions.     

Evidence for a vasopressin system in the rat heart

Traditionally, a hypothalamo-neurohypophysial system is thought to be the exclusive source of arginine vasopressin (AVP), a potent antidiuretic, vasoconstricting, and growth-stimulating neuropeptide. We have identified de novo synthesis of AVP in the heart as well as release of the hormone into the cardiac effluents. Specifically, molecular cloning of sequence tags amplified from isolated, buffer-perfused, and pressure-overloaded rat hearts allowed the detection of cardiac AVP mRNA. Subsequent experiments revealed a prominent induction of AVP mRNA (peak at 120 minutes, 59-fold, P<0. 01 versus baseline) and peptide (peak at 120 minutes, 11-fold, P<0. 01 versus baseline) in these isolated hearts. Newly induced vasopressin peptide was localized most prominently to endothelial cells and vascular smooth muscle cells of arterioles and perivascular tissue using immunohistochemistry. In addition to pressure overload, nitric oxide (NO) participated in these alterations, because inhibition of NO synthase by Nomega-nitro-L-arginine methyl ester markedly depressed cardiac AVP mRNA and peptide induction. Immediate cardiac effects related to cardiac AVP induction in isolated, perfused, pressure-overloaded hearts appeared to be coronary vasoconstriction and impaired relaxation. These functional changes were observed in parallel with AVP induction and largely prevented by addition of a V1 receptor blocker (10(-8) mol/L [deamino-Pen1, O-Me-Tyr2, Arg8]-vasopressin) to the perfusion buffer. Even more interesting, pressure-overloaded, isolated hearts released the peptide into the coronary effluents, offering the potential for systemic actions of AVP from cardiac origin. We conclude that the heart, stressed by acute pressure overload or NO, expresses vasopressin in concentrations sufficient to cause local and potentially systemic effects.     

Expression of monocyte chemoattractant protein 1 mRNA in human idiopathic pulmonary fibrosis.

Macrophages are thought to play an important role in the pathologic changes associated with idiopathic pulmonary fibrosis (IPF). The mechanisms for increased monocyte/macrophage recruitment in IPF are unknown. Monocyte chemoattractant protein 1 (MCP-1) is the predominant monocyte chemoattractant secreted by a variety of different cell types in culture. We examined the expression of MCP-1 mRNA and its protein product in vivo in IPF and non-IPF lung specimens by in situ hybridization and immunocytochemistry. The cell types expressing MCP-1 in vivo were identified by immunostaining with specific antibodies. We demonstrated the expression of MCP-1 mRNA in pulmonary epithelial cells, in monocytes/macrophages, and in vascular endothelial and smooth muscle cells. Lung epithelial cells in patients with IPF strongly expressed MCP-1 mRNA and its protein product. In contrast, epithelial cells in non-IPF specimens did not express MCP-1 mRNA. Macrophages and vascular endothelial and smooth muscle cells were shown to express MCP-1 in both IPF and non-IPF lung specimens. These findings provide a basis for the understanding of the in vivo physiologic processes that mediate monocyte/macrophage recruitment and infiltration in the lung interstitium and the pathologic state contributing to an increased alveolar monocyte/macrophage population and inflammation in IPF.     

Shear stress induces angiotensin converting enzyme expression in cultured smooth muscle cells: possible involvement of bFGF

OBJECTIVE: Hemodynamic stresses are considered to be important regulators of gene expression in vascular cells. In this study, we have investigated the role of shear stress on ACE expression in cultured rat vascular cells, and focused on the regulation of ACE expression in smooth muscle cells. METHODS: Rat aortic endothelial cells, smooth muscle cells and fibroblasts isolated from Wistar rats were submitted to shear stress using a laminar shear flow parallel chamber. RESULTS: A 10 dynes/cm2 shear rate for 24 h increased ACE activity in the three vascular cell types (x 2.14 in endothelial cells, x 2.9 in smooth muscle cells, x 3.33 in fibroblasts). This induction was blocked by a 24 h pre-incubation with a translation blocker (10(-4)M cycloheximide) showing the role of protein neosynthesis. Therefore the study was focused on smooth muscle cells and we demonstrated that the increase in ACE activity was due to an elevation in ACE mRNA level in response to a 10 dynes/cm2 shear stress for 24 h. This induction was dependent on the shear intensity (P < 0.0001). Six hours of a 15 dynes/cm2 shear stress showed no effect on ACE activity or mRNA expression. In contrast, the same duration of shear significantly increased bFGF mRNA level (x 3.7). Conversely, bFGF dose dependently increased ACE mRNA expression and activity in smooth muscle cells. This result suggests that bFGF could be one of the potential inductors of ACE expression in the stressed smooth muscle cells. CONCLUSIONS: Mechanical stress increases ACE expression in vascular cells. bFGF could be one of the potential factors involved in this activation. This phenomenon could participate in the role of ACE activity in vascular wall remodeling.     

Endogenous thrombospondin 1 protects the pressure-overloaded myocardium by modulating fibroblast phenotype and matrix metabolism

The matricellular protein thrombospondin (TSP) 1 is induced after tissue injury and may regulate reparative responses by activating transforming growth factor-β, by suppressing angiogenesis and by modulating inflammation and matrix metabolism. We hypothesized that endogenous TSP-1 may be involved in the pathogenesis of cardiac remodeling in the pressure-overloaded heart. Myocardial TSP-1 expression was increased in a mouse model of pressure overload because of transverse aortic constriction. TSP-1(-/-) mice exhibited increased early hypertrophy and enhanced late dilation in response to pressure overload. Pressure-overloaded TSP-1 null mice had intense degenerative cardiomyocyte changes, exhibiting more extensive sarcomeric loss and sarcolemmal disruption when compared with wild-type hearts. Accentuated hypertrophy and cardiomyocyte injury in TSP-1(-/-) hearts was accompanied by increased myofibroblast density. However, despite a 2-fold higher infiltration of the cardiac interstitium with myofibroblasts, pressure-overloaded TSP-1 null hearts did not exhibit significantly increased collagen content when compared with wild-type hearts. The disproportionately low collagen content in TSP-1 null hearts was attributed to infiltration with abundant, but functionally defective, fibroblasts that exhibited impaired myofibroblast differentiation and reduced collagen expression in comparison with wild-type fibroblasts. Impaired myofibroblast activation in TSP-1 null hearts was associated with reduced Smad2 phosphorylation reflecting defective transforming growth factor-β signaling. Moreover, TSP-1 null hearts had increased myocardial matrix metalloproteinase 3 expression and enhanced matrix metalloproteinase 9 activation after pressure overload. TSP-1 upregulation in the pressure-overloaded heart critically regulates fibroblast phenotype and matrix remodeling by activating transforming growth factor-β signaling and by promoting matrix preservation, thus preventing chamber dilation.     

Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype

We previously reported that adenosine A2B receptor activation stimulates angiogenesis. Because hypoxia is a potent stimulus for the release of both adenosine and angiogenic factors, we tested the hypothesis that hypoxia alters the expression of adenosine receptors toward an "angiogenic" phenotype. We used human umbilical vein endothelial cells (HUVECs) and bronchial smooth muscle cells (BSMCs) because, under normoxic conditions, adenosine does not release vascular endothelial growth factor (VEGF). HUVECs expressed a characteristic A2A phenotype (the selective A2A agonist CGS21680 was as potent as the nonselective agonist 5'-N-ethylcarboxamidoadenosine [NECA] in generating cAMP). Hypoxia (4.6% O2, 3 hours) decreased A2A mRNA from 1.56+/-0.3% to 0.16+/-0.01% of beta-actin expression but increased A2B mRNA from 0.08+/-0.01% to 0.27+/-0.05%. Consistent with changes in receptor expression, CGS21680 failed to increase cAMP in hypoxic HUVECs, whereas NECA remained active (A2B phenotype), and NECA increased VEGF release from 9.5+/-1.0 to 14.2+/-1.2 pg/mL (P<0.05), indicating that increased A2B receptors were functionally coupled to upregulation of VEGF. Hypoxia had similar effects on BSMCs, increasing A2B mRNA by 2.4+/-0.3-fold, from 0.42+/-0.04% to 1.00+/-0.13% of beta-actin. Whereas NECA had no effect on VEGF release in normoxic BSMCs, it increased VEGF release in hypoxic BSMCs, from 74.6+/-9.6 to 188.3+/-16.7 pg/mL (P<0.01), and a selective A2B antagonist, CVT-6694, inhibited this increase. A2B receptors activated a VEGF reporter made unresponsive to hypoxia by mutating its hypoxia-inducible factor-1 (HIF-1) binding element, indicating a mechanism independent of HIF-1. In conclusion, hypoxia modulates the expression of adenosine receptors in human endothelial and smooth muscle cells toward an A2B"angiogenic" phenotype.     

Nitric oxide impacts endothelin-1 gene expression in intrapulmonary arteries of chronically hypoxic rats.

This study aimed to investigate whether nitric oxide (NO) could inhibit the elevated endothelin-1 (ET-1) gene expression by pulmonary artery endothelial cells or smooth muscle cells in chronically hypoxic rats by use of in situ hybridization. Male Wistar rats (n = 40) were randomly divided into 1-week hypoxia group, 1-week hypoxia with L-arginine (L-arg) group, 1-week hypoxia with N(omega)-nitro-L-arginine methyl ester (L-NAME) group, 2-week hypoxia group, 2-week hypoxia with L-arg group, and 2-week hypoxia with L-NAME group. All rats were put into a normobaric hypoxic chamber with an oxygen concentration of 10 +/- 0.5% for hypoxic challenge. The results showed that most pulmonary arteries had 1-50% of the endothelial cells showing positive signals for ET-1 expression in hypoxic rats, which was significantly suppressed by L-arg. L-NAME, however, significantly augmented ET-1 gene expression in pulmonary artery endothelial cells and smooth muscle cells. The results suggest that endogenous NO markedly inhibits ET-1 mRNA expression in both pulmonary artery endothelial cells and smooth muscle cells in chronically hypoxic rats, which may be one of the mechanisms by which NO modulates hypoxic pulmonary circulation.     

Increased expression and function of the myocardial Na–K–2Cl cotransporter in failing rat hearts

Abstract Recent studies indicate a role of the Na–K–2Cl cotransporter (NKCC) in regulation of myocardial function. However, potential pathophysiological properties of NKCC in conditions like myocardial infarction (MI) and heart failure have not been explored. We investigated the cellular localization of myocardial NKCC and whether myocardial NKCC levels are changed upon induction of post-infarction heart failure in rats. Immunohistochemical analysis demonstrated extensive distribution of NKCC in normal rat myocardium with fairly strong expression in cardiomyocytes, fibroblasts, vascular endothelial cells, as well as smooth muscle cells. Myocardial mRNA levels of NKCC were investigated at 2, 7 and 28 days after induction of MI or sham operation, but no changes were found. Cardiomyocytes and non-cardiomyocytes were isolated 7 days after induction of MI or sham operation. An ∼2-fold increase of the NKCC mRNA levels was found in isolated cardiomyocytes from heart failure rats compared to that of sham-operated rats (P < 0.001), whereas a trend towards decreased mRNA levels of NKCC in isolated non-cardiomyocytes was observed. In addition, we found a bumetanide sensitive ⁸⁶Rb⁺ influx mechanism present in the hearts after induction of MI (P < 0.05). Thus, our data indicate cardiomyocyte specific increase in NKCC mRNA levels and increased NKCC activity in post-infarction heart failure. Our results may indicate a potential role of NKCC during post-infarction remodeling.     

Short- and long-term interactions of endothelium and vascular smooth muscle in coculture: effects on cyclic GMP production

In intact blood vessels, many vasodilators act by stimulating the release from endothelium of factor(s) that relax vascular smooth muscle and stimulate increases in cGMP. To investigate how endothelium regulates cGMP production in vascular smooth muscle, bovine aortic endothelial cells and rat aortic smooth muscle cells were cultured both separately and together in cocultures for 48 hr. Nitroprusside (1 mM) increased intracellular cGMP concentration 30-fold in smooth muscle cells (from a basal level of 103 +/- 54 fmol/mg of cell protein to 2920 +/- 1800 fmol/mg) but only 2-fold in endothelial cells (from 41 +/- 7 fmol/mg to 93 +/- 23 fmol/mg). When endothelial and smooth muscle cells were cocultured as a mixed cell population (1:1 cell ratio), both basal and nitroprusside-stimulated cGMP levels were significantly increased (550 +/- 250 and 13,240 +/- 9950 fmol/mg of total cell protein, respectively). The calcium ionophore A23187 (10 microM) caused no increase in cGMP concentration in either cell type cultured alone but produced a 6-fold increase in cocultures. Neither aspirin nor 5,8,11,14-icosatetraynoic acid influenced these results. No changes in cAMP levels were detected. Using cocultures in which one cell type was grown on microcarrier beads, we have shown that cGMP increased only in vascular smooth muscle cells and was not dependent upon the formation of junctions between endothelium and smooth muscle cells. In long-term (48-hr) mixed-cell cocultures, but not in short-term microcarrier cocultures, amplification of the nitroprusside-induced increase in cGMP was observed. These results show that responses associated with endothelium-dependent relaxation can be reconstituted in cultured endothelial and vascular smooth muscle cells and that endothelium generates a humoral factor(s) that stimulates accumulation of smooth muscle cGMP and has a longer-term effect that amplifies guanylate cyclase stimulation by nitroprusside, a drug acting directly upon smooth muscle to stimulate formation of the cyclic nucleotide. Cultured cells provide a valuable model system for the study of endothelium-vascular smooth muscle interactions.     

Repetitive myocardial stunning in pigs is associated with the increased expression of inducible and constitutive nitric oxide synthases

OBJECTIVES: Nitric oxide (NO) has complex effects on myocardial function particularly following ischaemia-reperfusion. The goal of this study was to examine the result of repetitive myocardial stunning on myocardial NO release and expression of inducible (iNOS) and constitutive (eNOS) NO synthases. METHODS AND RESULTS: Propofol anaesthetised pigs underwent ten, 2-min episodes of circumflex artery occlusion (n = 6) or acted as sham operated controls (n = 4). Measurements of segment shortening demonstrated a fall in function in the ischaemic territory to 52.5 +/- 7.3% (mean +/- S.E.M.) of baseline shortening 30 min after the stunning stimulus, recovering to 92 +/- 8.7% 5.5 h later. Function remained stable in sham controls. The change in venous-arterial [NO] between baseline and 6 h reperfusion was found to be significantly different between the two groups (0.2 +/- 0.7 in stunned vs. -4.3 +/- 1.6 microM in shams; P < 0.02). Western blotting and band optical density used to compare tissue from stunned territory (S), non-stunned territory (IC) and sham control animals (SC) demonstrated this was associated with an increase in the expression of both iNOS (S: 93 +/- 13.4, IC: 37 +/- 2.4 and SC: 25 +/- 4 [arbitrary units], P < 0.01 and P = 0.031) and eNOS (S: 104 +/- 7.4, IC; 62.5 +/- 7.4 and SC; 75.7 +/- 0.6, P < 0.03 and P < 0.01) in stunned myocardium. Immunocytochemistry localised iNOS reactivity to vascular smooth muscle cells and cardiomyocytes in stunned tissue and eNOS reactivity to endothelial cells. CONCLUSION: Recovery from repetitive myocardial stunning is associated with the increased expression of both iNOS and eNOS and would be compatible with a protective role for both these enzymes. This finding has possible relevance for both the late window of ischaemic preconditioning and myocardial hibernation.     

高糖对人大血管细胞钾依赖钠钙交换蛋白6表达的影响

目的 观察钾依赖钠钙交换蛋白6（NCKX6）基因在人主动脉内皮细胞、平滑肌细胞和巨噬细胞的表达，以及高糖对大血管NCKX6表达的影响。方法 Western印迹检测正常血管组织和2型糖尿病血管组织NCKX6蛋白的差异表达，RT—PCR检测NCKX6基因在人主动脉平滑肌细胞（HASMC）、人主动脉内皮细胞（HAEC）和人血管巨噬细胞（THP-1）中的表达，Northern印迹杂交检测高糖作用HAEC细胞NCKX6mRNA的表达差异。结果 Western印迹初步显示NCKX6在2例糖尿病患者大血管中明显高表达，RT—PCR观察到NCKX6基因主要在HAEC细胞中表达，而HAMSC和THP-1细胞中表达量低。Northern印迹杂交进一步观察到随着培养液D-葡萄糖浓度的增加，作用72h后HAEC细胞NCKX6mRNA表达明显增高，30mmol／LD-葡萄糖作用下NCKX6mRNA表达量可增加5—6倍。结论 NCKX6主要在人主动脉内皮细胞中表达，并且受到葡萄糖的调控。提示NCKX6与糖尿病大血管病变有密切的关系。     

Increased vascular endothelial growth factor expression in human hearts with microvascular fibrin

We have shown that microvascular changes that promote fibrin deposition in human cardiac allografts adversely affect clinical outcome. However, some allografts exhibit phenotypic changes in capillaries following the deposition of fibrin, which subsequently provide a significant survival advantage. The mechanism(s) involved in these capillary changes is(are) unknown. Similarly, although we have shown a significant temporal relationship between microvascular fibrin deposition and vascular endothelial growth factor (VEGF) immunoreactivity in cardiac allografts, the cellular source and relative changes in VEGF gene expression under these conditions are not known. Using immunocytochemical techniques, biopsies devoid of fibrin deposition lacked detectable VEGF immunoreactivity, whereas biopsies with fibrin deposition showed VEGF immunoreactivity in cardiocytes, interstitium, and some microvessels. By in situ hybridization, biopsies without microvascular fibrin deposition showed faint VEGF hybridization signals confined primarily to cardiocytes. In biopsies with fibrin deposition, strong VEGF hybridization signals were detected in cardiocytes, arteriolar smooth muscle cells were occasionally labeled, and endothelial cells were rarely labeled. By quantitative RT-PCR, biopsies with fibrin deposition (n=5) relatively expressed approximately three-fold more VEGF mRNA than biopsies without fibrin deposition (n=5 P=0.02). Serum VEGF titers also were greater (P=0.01) in recipients with fibrin deposition (372.9+/-66.7 pg/ml n=18) compared to recipients without fibrin deposition (172.1+/-25.0 pg/ml n=16). Collectively, these results support the hypothesis that increased myocyte-derived VEGF production following microvascular fibrin deposition in transplanted human hearts may act in a paracrine manner to promote activational and phenotypic changes in capillaries that provide a survival advantage for the allografts.     

Immunohistochemical identification of cell types in normal and in bleomycin-induced fibrotic rat lung. Cellular origins of interstitial cells

There is substantial tissue reorganization in the interstitium in pulmonary fibrosis. In order to determine the origin of cells in the interstitium of normal or fibrotic rat lung, we stained lung tissue immunohistochemically with antiserums directed against intermediate filament proteins specific for cells of mesenchymal or muscle origin. Vimentin, specific for mesenchymal cells, was isolated from cultured 3T3 fibroblasts, and desmin, specific for muscle cells, was isolated from chicken gizzard and antiserums prepared in guinea pigs. Paraffin-embedded sections of normal or fibrotic rat lung from rats 28 days after treatment with bleomycin sulfate were reacted with each antiserum and stained by the peroxidase-antiperoxidase technique. Antivimentin antiserum stained discrete interstitial cells of both control and fibrotic lung. Smooth muscle surrounding large airways and vascular smooth muscle was not significantly stained with this antiserum but was heavily stained with antidesmin. However, antidesmin did not stain cells in the interstitium of normal or fibrotic rat lung. These results suggest that most cells in the normal as well as in the fibrotic interstitium, including contractile interstitial cells, are probably of mesenchymal (fibroblastic) rather than of smooth muscle origin.     

Arteriolar occlusion causes independent cellular responses in endothelium and smooth muscle

OBJECTIVES: To test the hypothesis that arteriolar occlusion causes different cellular changes in endothelial and smooth muscle cells. METHODS: Cheek pouch arterioles (resting diameter 41 +/- 2 microm) of anesthetized hamsters were occluded briefly (<60 seconds) either upstream or downstream from an observation site. Changes in membrane potential and intracellular calcium concentration ([Ca(2+)](i)) of the endothelial or smooth muscle cells were determined by using fluorescence microscopy (ratiometric analysis). RESULTS: The pressure in the occluded segment decreased by 17.4 +/- 2.6 cm H(2)O during upstream occlusion and increased by 16.8 +/- 6 cm H(2)O during downstream occlusion (n = 5). Upstream occlusion caused vasoconstriction of the occluded segment by 2.4 +/- 0.4 microm, whereas downstream occlusion produced brief vasodilatation by 1.1 +/- 0.2 microm. The endothelial cells hyperpolarized during upstream or downstream occlusion (ratio change: 2.26 +/- 0.24% and 2.39 +/- 0.42%, respectively; p < 0.01, n = 5). There were no changes in endothelial [Ca(2+)](i). The smooth muscle cells depolarized (ratio change: -2.08 +/- 0.14%, n = 5) with an increase in [Ca(2+)](i) (ratio change: 2.92 +/- 0.16%, n = 6) during downstream occlusion. However, there was no detectable change in membrane potential or [Ca(2+)](i) of smooth muscle cells during upstream occlusion. All the changes rapidly recovered when occlusion was released. Responses of an in-situ isolated segment on a side branch revealed conducted dilatory signals caused by the occlusions. CONCLUSIONS: Our results show that the endothelial and smooth muscle cells respond independently to arteriolar occlusion. The endothelial and smooth muscle cells do not effectively communicate in [Ca(2+)](i) or membrane potential during acute arteriolar occlusion. Hyperpolarizing signals in endothelium cause conducted dilation.