Stimulation of DNA synthesis, activation of mitogen-activated protein kinase ERK2 and nuclear accumulation of c-fos in human aortic smooth muscle cells by ketamine.

Proliferation of vascular smooth muscle cells is known to be regulated by autocrine and paracrine stimuli, including extracellular matrix, reactive oxygen species, lipids, and biomechanical forces. The effect of many pharmacological agents to which smooth muscle cells may be exposed, however, is widely unexplored. Ketamine, an intravenous anaesthetic and a phencyclidine derivative, regulates diverse intracellular signalling pathways in smooth muscle cells, several of which are known to affect cell proliferation. The effect of ketamine on proliferative response of smooth muscle cells, however, is not determined. We tested the hypothesis that ketamine may regulate proliferation of smooth muscle cells, and investigated the effects of pharmacological doses of ketamine on their proliferative capacity by measuring DNA synthesis and activation of mitogen-activated protein (MAP) kinase signalling pathway in human aortic smooth muscle cells. DNA synthesis, as determined by incorporation of 3H-thymidine into DNA, was enhanced by 73% (P < 0.0001) and 130% (P < 0.0001) with 10 and 100 microm ketamine, respectively. Ketamine-induced DNA synthesis was dependent on de novo protein synthesis, as it was abolished by an inhibitor of protein synthesis, cycloheximide. A synthetic inhibitor of MAP kinase pathway, PD98059, decreased 50% (P < 0.0001) of ketamine-induced DNA synthesis, suggesting that the activation of MAP kinase pathway was partially responsible for ketamine-induced effects. Consistently, in-gel kinase assay and in vitro kinase assay of cell lysates showed ketamine-induced MAP kinase activation and expression of ERK2 (extracellular signal-regulated kinase) in smooth muscle cells. This effect of ketamine was not dependent on de novo protein synthesis. Immunofluorescent light microscopy showed ketamine-induced nuclear accumulation of c-fos, a downstream effect of MAP kinase activation, in smooth muscle cells. In conclusion, these data support the hypothesis of the study and demonstrate that ketamine, by stimulating DNA synthesis in human aortic smooth muscle cells, may have an impact on proliferative capacity of these cells. The present results also demonstrate that ketamine induces the activation of MAP kinase pathway and nuclear accumulation of transcription factor c-fos in smooth muscle cells. They further demonstrate that the activation of MAP kinases is partially responsible for ketamine-induced DNA synthesis in human aortic smooth muscle cells. Together, these findings suggest that ketamine may play a role as a pharmacological regulator of mechanisms involved in proliferation of smooth muscle cells.

Release of pro- and anti-angiogenic factors by human cardiac fibroblasts: effects on DNA synthesis and protection under hypoxia in human endothelial cells.

Myocardium consists of diverse cell types suggesting a role for cell-cell interaction in maintaining the structural and functional integrity of the heart. Cardiac fibroblasts are the source of extracellular matrix, growth factors and cytokines in the heart and their interactions with cardiac myocytes are recognized. Their effects on biological responses of endothelial cells, however, are vastly unexplored. Proliferation of endothelial cells is an essential stage of angiogenesis and contributes to development of coronary collaterals. This study was designed to evaluate the effect of soluble factors produced by cardiac fibroblasts on endothelial cell proliferation. Human cardiac fibroblast-conditioned medium (CF-CM) caused a significant increase (47%, P < 0.0001) in DNA synthesis in human umbilical vein endothelial cells (HUVEC), as determined by [(3)H]thymidine incorporation. This effect was dependent on de novo protein synthesis and activation of MAP kinases. Consistently, CF-CM induced the expression and activation of ERK2 in HUVEC. The CF-CM from which heparin-binding proteins were removed, had a significantly enhanced stimulatory effect on DNA synthesis in HUVEC compared to that of 'whole CF-CM'. Western analysis showed the presence of VEGF, bFGF, PDGF, TGF-beta(1), fibronectin and thrombospondin-1 in whole CF-CM. The individual immunodepletion of each factor from whole CF-CM showed that all were necessary for full activity of CF-CM. CF-CM caused a significant reversal of hypoxia-induced inhibition of DNA synthesis and enhanced expression of survival-associated protein, Bcl(2), in HUVEC. Together, these data show that cardiac fibroblasts release inhibitory and stimulatory factors, the net effect of which is an enhancement of DNA synthesis in endothelial cells. These results point to the role that cardiac fibroblasts may play in angiogenesis in the heart.

Gender-related differences in proliferative response of cardiac fibroblasts to hypoxia: effects of estrogen.

Ischemic heart disease is more prevalent in men than in women. The remodeling of extracellular matrix, is a structural correlate of heart failure of ischemic origin and proliferation of cardiac fibroblasts is a key factor in this remodeling. We asked if proliferative response of male and female cardiac fibroblasts is differentially susceptible to hypoxia. DNA synthesis, using 3H-thymidine incorporation was compared under hypoxia (2% O2) in cardiac fibroblasts obtained from adult, age-matched male and female rat heart. In female cells DNA synthesis remained unchanged under hypoxia and this resistance was dependent on tyrosine kinase activation, as it was abolished in the presence of genistein, a tyrosine kinase inhibitor. Male cells, on the other hand, were susceptible to hypoxia and their DNA synthesis was reduced significantly (70%, (p < 0.0001). This effect was partially reversed by inhibition of tyrosine kinase. Western analysis showed a higher abundance of tyrosine phosphorylated proteins in male cells compared to female cells as well as differences in molecular weight of basal and hypoxia-induced tyrosine-phosphorylated proteins between male and female cells. The presence of estrogen (17-beta estradiol, 10 nM) altered the response of both cells to hypoxia. In female cells the combined effect of hypoxia and estrogen led to inhibition of DNA synthesis, whereas in male cells estrogen partially reversed the hypoxia-induced inhibition of DNA synthesis (37% (p < 0.01) inhibition in the presence of estrogen vs. 70% (p < 0.0001) inhibition in the absence of estrogen). The effects of estrogen in male and female cells were mediated via estrogen receptors as they were reversed by the pure anti-estrogen, ICI 182,780. Western analysis of cell lysate showed hypoxia-induced increase in the level of estrogen receptor beta in both male and female cells. Gel shift analysis showed hypoxia-induced increase in cytoplasmic ERE (estrogen response element)-binding activity and decrease in nuclear ERE-binding in male cells. In female cells cytoplasmic and nuclear ERE-binding activities remained unchanged under hypoxia. Together, these data demonstrate that while female cells are resistant to hypoxia-induced inhibition in DNA synthesis, male cells are susceptible; intracellular pathways involving tyrosine phosphorylation are involved in the response of both cells; and estrogen, via estrogen-receptor-dependent mechanisms, differentially alters the response of male and female cells to hypoxia.

Estrogen enhances proliferative capacity of cardiac fibroblasts by estrogen receptor- and mitogen-activated protein kinase-dependent pathways.

The role of female hormones in the prevalence of cardiac diseases are recognized but not fully explored. Proliferation of cardiac fibroblasts, the cellular origin of the extracellular matrix proteins, growth factors and cytokines in the heart, is an important underlying mechanism in the pathophysiological remodeling of the myocardium. In this study, we have investigated the effect of estrogen (17 beta-estradiol) on proliferative capacity of cardiac fibroblasts obtained from adult female rat heart. DNA synthesis, as determined by incorporation of 3H-thymidine into DNA, increased in estrogen-treated cells. In the presence of tamoxifen, an anti-estrogen with high affinity for estrogen receptor. 17 beta-estradiol-induced stimulation of DNA synthesis was abolished. Alpha-estradiol, a stereo-isomer which does not bind the estrogen receptor, did not change DNA synthesis. In the presence of a synthetic inhibitor of MAP kinase pathway. PD98059, estrogen failed to stimulate DNA synthesis. In-gel kinase assays showed rapid and transient increased phosphorylation of MAP kinase substrate, myelin basic protein (MBP), at 42 and 44 kDa by 17 beta-estradiol, which was not observed in the presence of PD98059 and tamoxifen, not induced by alpha-estradiol and persisted in the absence of protein kinase C. In vitro kinase assay confirmed 17 beta-estradiol-induced activation of ERK1 and ERK2, with predominant effect on ERK2 in cardiac fibroblasts. The results of immunofluorescent light microscopy using anti-type alpha and beta estrogen receptor antibodies showed the expression of estrogen receptor types alpha and beta in control untreated cells, and indicated that type beta receptor is the predominant type with both cytoplasmic and nuclear localization. 17 beta-estradiol treatment of cardiac fibroblasts induced the translocation of receptor protein to the nuclei. Together, these data provide evidence that cardiac fibroblasts are cellular targets for direct effects of estrogen, and that this hormone enhances proliferative capacity of cardiac fibroblasts via estrogen receptor- and MAP kinase-dependent mechanisms. These data further suggest that estrogen, by its growth-enhancing effects in cardiac fibroblasts, can regulate the remodeling of the extracellular matrix and alter the microenvironment of cardiac cells, and hence exert an impact on the integrity of myocardial function.

An activator protein-1 (AP-1) response element on pro alpha1(l) collagen gene is necessary for thyroid hormone-induced inhibition of promoter activity in cardiac fibroblasts.

Thyroid hormone-induced ventricular hypertrophy is characterized by the absence of fibrosis. Previously, we demonstrated that thyroid hormone inhibits collagen type I gene expression in the myocardium and in cardiac fibroblasts. We also demonstrated that thyroid hormones act as inhibitor of pro alpha1(l) collagen promoter activity. In this study we determined the sequences on pro alpha1(l) collagen gene and transcription factors in cardiac fibroblasts involved in the inhibitory effect of 3,3',5-triiodothyronine (T3). Transient transfection of cells with chloramphenicol acetyl transferase (CAT)-linked deletion mutants of pro alpha1(l) collagen promoter demonstrated that the inhibitory effect of T3 is transmitted via proximal sequences(-225/+115). Gel shift analysis using [32P]-labeled -225/+115 gene fragment and nuclear proteins of cardiac fibroblasts showed T3-induced DNA binding by two proteins. Analysis of non-overlapping restriction sub-fragments by gel shift along with supershift analysis with antibodies to types alpha and beta thyroid hormone receptors identified the lower molecular weight DNA-binding protein as beta receptor and confirmed that the T3-induced protein-DNA binding sites are located at -15/+115. Selective base mutation (C in place of G at +93 and G in place of C at +97) in the activator protein-1 (AP-1) core binding motif(+92/+97) abolished the higher molecular weight T3-induced DNA-protein complex obtained with [32P]-labeled wild type sequences (-225/+115). Additional gel shift analyses using an oligonucleotide containing the AP-1 core binding motif, as an unlabeled competitor and as [32P]-labeled probe, confirmed the T3-induced protein binding to an AP-1 site. Transient transfection with CAT-linked -225/+115 sequences in which the AP-1 site was mutated abolished the T3-induced inhibition of CAT activity. Together, these findings identify sequences necessary for T3-induced inhibition of collagen type I promoter to which thyroid hormone receptor type beta and protein(s) with affinity for AP-1 element bind. They also demonstrate that the AP-1 response element located on these sequences is necessary for T3-induced inhibition of pro alpha1(l) collagen promoter activity. These data identify molecular mechanisms involved in thyroid hormone-induced inhibition of collagen expression in the heart.

Hypoxia regulates basal and induced DNA synthesis and collagen type I production in human cardiac fibroblasts: effects of transforming growth factor-beta1, thyroid hormone, angiotensin II and basic fibroblast growth factor.

Analysis of post-infarct ventricular remodeling consistently shows the accumulation of collagen in failing heart. The goal of this study was to gain insights into the underlying mechanisms of this event. We determined the effect of hypoxia, caused as the result of ischemia, on biological responses including cell viability, basal and growth factor-stimulated proliferative capacity and collagen type I production in cardiac fibroblasts obtained from adult human heart. The cell viability, as examined by light microscopy and analysis of DNA, did not change by hypoxia (2% oxygen). Basal level of protein synthesis, as determined by measuring the incorporation of 3H-leucine, decreased (30%, P<0.05) under hypoxia. Transforming growth factor-beta (TGF-beta1)- and thyroid hormone (T3)-induced increases in protein synthesis did not change under hypoxia. In contrast, basic fibroblast growth factor (bFGF)-stimulated protein synthesis enhanced significantly under hypoxia. Angiotensin II (Ang II)-treatment, which did not induce significant changes in protein synthesis under ambient conditions, led to moderate but significant increase under hypoxia. Basal level of DNA synthesis, as determined by measuring the incorporation of 3H-thymidine into DNA, decreased (32%, P<0.05) under hypoxia. The TGF-beta1-induced inhibition of DNA synthesis which was observed under ambient conditions was reversed [61% (P<0.005) increase under hypoxia]. Under ambient conditions, T3, Ang II and bFGF stimulated DNA synthesis and their effects were enhanced under hypoxia. Northern analysis showed a 46% (P<0.05) increase in the level of pro alpha1(l) collagen mRNA under hypoxia. The TGF-beta1-induced increase in the level of pro alpha1(l) collagen mRNA, under ambient conditions, was not observed under hypoxia. On the other hand, the T3-induced decrease in pro alpha1(l) collagen mRNA was reversed under hypoxia. Ang II- and bFGF-treatment of human cardiac fibroblasts did not cause detectable changes in the level of pro alpha1(l) collagen mRNA under ambient conditions or hypoxia. At the protein level, the amount of immunoreactive collagen type I, as determined by immunoslot blot analysis, was increased (33%, P<0.05) under hypoxia. Treatment of human cardiac fibroblasts with TGF-beta1 and T3 under ambient conditions led to diminished level of collagen type I. Under hypoxia, however, effect of both factors was reversed. The level of immunoreactive collagen type I in Ang II- and bFGF-treated cells, which was comparable to that in untreated cells under ambient conditions, remained unchanged under hypoxia. Together, these results provide evidence that hypoxia regulates growth, proliferative capacity and collagen type I production in human cardiac fibroblasts, and that although hypoxia alone may not be a stimulus for human cardiac fibroblast proliferation, it enhances growth factor-induced DNA synthesis in those cells. Furthermore, hypoxia by increasing the basal levels of collagen type I and by reversing the TGF-beta1- and T3-induced inhibition of collagen type I gene expression in human cardiac fibroblasts can enhance overall collagen type I production. Combinatorial effects of hypoxia on proliferation and collagen type I production in cardiac fibroblasts contribute to the post-infarct remodeling of the collagen matrix in failing human heart.

A simple method for preparation of cultured cardiac fibroblasts from adult human ventricular tissue.

Cardiac fibroblasts constitute greater than 90% of non-myocyte cells in the heart. Because they are responsible for synthesis of components of the extracellular matrix, growth factors and cytokines in the myocardium, they play an important role in normal and pathologic performance of the heart. An understanding of their biology requires in depth studies in a stable and reliable system in which the biological responses of cardiac fibroblasts to various stimuli can be determined. With the exception of few, all studies have been performed on cardiac fibroblasts obtained from rodent hearts. We present a method for isolation and subsequent culture of viable cardiac fibroblasts from ventricular tissue of adult human. This method allows rapid and reliable isolation and subsequent culture of cardiac fibroblasts from adult heart tissue without the need for cumbersome isolation techniques and complex nutrient-enriched and hormone-supplemented culture media for maintenance.

Characterization of adult human heart fibroblasts in culture: a comparative study of growth, proliferation and collagen production in human and rabbit cardiac fibroblasts and their response to transforming growth factor-beta1.

Fibroblasts constitute the majority of non-myocyte cells in the heart, but little is known about the biology of human cardiac fibroblasts. We have cultured and characterized fibroblasts from young adult human heart and compared their biological features with those of cells obtained from the ventricular tissue of sex- and developmental-stage-matched rabbits. Cell morphology and growth was determined by light microscopy and by measuring protein synthesis. The proliferative capacity of cells was determined by measuring the doubling time and the level of incorporation of 3H-thymidine into DNA under normal conditions of cell culture and in response to several growth factors. Production of collagen type I was determined at the mRNA level by assessing the steady state level of pro alpha1 20(l) collagen mRNA in cardiac fibroblasts under normal culture conditions and in response to transforming growth factor-beta (TGF-beta), and at the protein level by immuno-slot blot analysis. Ventricular tissue from adult human heart was used successfully for the preparation of passagable (up to nine passages) cultured cardiac fibroblasts. They had a significantly larger surface area per cell than rabbit cells. Under normal culture conditions, they showed a lower rate of DNA synthesis and longer doubling time than rabbit cardiac fibroblasts at the matching passage. The magnitude of growth-factor-induced changes in the incorporation of 3H-thymidine into DNA was lower in human cells than in rabbit cardiac fibroblasts, as was the level of mRNA for pro alpha1(l) collagen. The TGF-beta1-induced increase in pro alpha1(l) collagen mRNA was modest in human cardiac fibroblasts. Thus, significant differences exist between the biological properties of human and rabbit cardiac fibroblasts in culture.

Regulation of proliferative response of cardiac fibroblasts by transforming growth factor-beta 1.

Cardiac fibroblasts constitute greater than 90% of the non-myocyte cells in the heart. Previously, it was established that cardiac fibroblasts are predisposed to transformation into a phenotype with muscle-specific features and that transforming growth factor-beta 1 (TGF-beta 1) is a specific inducer of this event. In this study the hypothesis that TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts is associated with their altered proliferative capacity is tested. Therefore the effects of TGF-beta 1 on DNA synthesis in cardiac fibroblasts under normal conditions of cell culture and in response to a potent mitogen, basic fibroblasts growth factor (bFGF) were determined. The results showed that TGF-beta 1 at 15 ng/ml (a concentration that induces fibroblast "transformation") had a regulatory effect on proliferative capacity of cardiac fibroblasts which varied as the function of cell density in culture. In subconfluent and confluent cultures, pre-treatment of cardiac fibroblasts with TGF-beta 1 for 24 h resulted in a dramatic shift in the bFGF-induced stimulation of DNA synthesis. TGF-beta 1-induced inhibition of DNA synthesis in cardiac fibroblasts coincided with their phenotypic modulation as evidenced by the expression of sarcomeric actin mRNA and morphological changes. Cross-linking studies with [125I]-labeled TGF-beta 1 showed the presence of conventional types I, II and III TGF-beta 1 receptor complexes on cardiac fibroblasts and their binding to TGF-beta 1 under the experimental conditions. In summary, these data indicate that the proliferative capacity of cardiac fibroblasts is controlled by TGF-beta 1. They further suggest that the TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts may be extended to include their altered proliferative capacity.

Upregulation of collagen type I gene expression in the ventricular myocardium of thyroidectomized male and female rats.

It is established that thyroxine-induced ventricular hypertrophy is associated with downregulation of collagen type I gene expression and increased collagen turnover in the ventricular tissue. The present study was undertaken to test the hypothesis that circulating thyroid hormones may have a regulatory impact on the biosynthesis of the collagen matrix in the heart. To this end, we determined collagen gene expression and deposition in the hearts of male and female Sprague-Dawley rats after surgical thyroidectomy. The serum levels of 3,3'5-triiodothyronine (T3) and 3,5,3',5'-tetraiodothyronine (T4) in thyroidectomized and age/sex-matched sham-operated rats were determined by radioimmunoassay and fluorescence analysis of the serum, respectively. On day 14 post-surgery, the plasma levels of both T3 and T4 in thyroidectomized rats were decreased by greater than 85% compared with those in matching sham-operated control rats. At this time, Northern analysis of ventricular RNA from thyroidectomized rats showed a 160% (P = 0.0079) increase for pro alpha 1 (I) and a 43% increase (P = 0.0484) for pro alpha 2 (I) collagen mRNAs in the ventricular tissue of male rats compared with that in the heart of age-matched, sham-operated control rats. In the female rats, thyroidectomy led to 63% (P = 0.0469) increase in the abundance of pro alpha 1 (I) collagen and 50% (P = 0.034) increase for pro alpha 2 (I) collagen in ventricular tissue. At the protein level, the amount of collagen type I as determined by immuno-slot blotting of ventricular homogenates, was increased in the ventricular tissue of both male (131%, P = 0.0371) and female (108%, P = 0.0464) rats. Comparison of the changes in males v females showed relatively greater increases in the level of collagen type I mRNA and protein in ventricular tissue of thyroidectomized males. Of particular note, were the increases in the immunoreactive TGF-beta in ventricular tissue of thyroidectomized male and female rats which showed a pattern similar to that of changes in collagen type I. Immunofluorescent light microscopy of frozen heart sections, showed significant remodeling of the type I collagen fibers in the ventricular myocardium of thyroidectomized rats compared with age/sex-matched sham-operated rat heart. Together, these findings suggest that circulating thyroid hormones play a role in physiological regulation of collagen type I biosynthesis in the heart and this role may vary in males and females. They further suggest that normal production of collagen matrix in the heart may be dependent on the functional status of thyroid hormones.

Regulation of mRNA transcripts and DNA synthesis in the rat heart following intravenous injection of transforming growth factor beta 1.

Transforming Growth Factor-beta 1 (TGF-beta 1) is expressed in the heart by muscle and non-muscle cardiac cells. In vitro, cardiac myocytes and non-muscle cells including cardiac fibroblasts and endothelial cells respond to regulatory effects of TGF-beta 1. Expression of TGF-beta 1 in the heart is subject to regulation by hemodynamic stimuli. Increased expression of mRNA transcripts for TGF-beta 1 has been reported in several models of cardiac hypertrophy. The objective of this study was to determine the effect of TGF-beta 1 in the myocardium. TGF-beta 1 was injected intravenously. Expression of mRNA transcripts for functional and structural proteins was determined by Northern hybridization analysis. DNA synthesis was determined by measurement of 3H-thymidine incorporation into ventricular DNA. The results showed differential regulation of mRNAs for myocyte- and non-myocyte-specific proteins in the heart of TGF-beta 1 treated rats. Moderate but statistically significant decrease in DNA synthesis was observed in the heart of TGF-beta 1 treated rats (37.5%, P < 0.025). Together, these data point to a physiological role for TGF-beta 1 in the heart. They further suggest that similar to its diverse in vitro cell-specific regulatory effects, TGF-beta 1 may have multicellular targets in the heart. Effect of TGF-beta 1 alone or combined with those of other cytokines/hormones that come into play, as the result of its administration, may be responsible for altered gene expression and DNA synthesis in the myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Nicotine regulates collagen gene expression, collagenase activity, and DNA synthesis in cultured cardiac fibroblasts.

Cardiac fibroblasts that reside in the interstitium are the cellular origin of collagen and other proteins of the extracellular matrix in the heart. We have previously shown that in vitro gene expression, proliferation and even phenotypic features of cardiac fibroblasts are subject to regulation by biological factors such as hormones, growth factors and neurotransmitters. The influence of nicotine, the active ingredient of tobacco, on risk factors for cardiac diseases is well known. In vivo adverse effects of nicotine are as the result of its direct and indirect effects. The cellular and molecular mechanisms of direct effects of nicotine in the heart are widely unknown. The objective of this study was to investigate if nicotine has direct influence on cardiac fibroblasts. To this end, we studied the effects of nicotine on cultured cardiac fibroblasts. Northern hybridization analysis of RNA extracted from cardiac fibroblasts, enzymography of conditioned medium of cardiac fibroblasts and [3H]-thymidine incorporation into DNA of cardiac fibroblasts were used to examine the effects of nicotine on collagen gene expression, collagenase activity and DNA synthesis respectively. Treatment of cardiac fibroblasts with nicotine (10 micrograms/ml) led to a 31% (P < 0.05) decrease in the abundance of mRNA for pro alpha 1(I) but not pro alpha 2(I) collagen compared with control untreated cells. Nicotine treatment of cardiac fibroblasts also led to decreased collagenase activity (62%, P < 0.001) in the conditioned medium of those cells in culture. Studies with [3H]-thymidine incorporation into DNA of cardiac fibroblasts showed a nicotine-induced decrease (39%, P < 0.001) in DNA synthesis in those cells.(ABSTRACT TRUNCATED AT 250 WORDS)