Shanhong Ling

EFFECTS OF A CHINESE HERBAL PREPARATION ON VASCULAR CELLS IN CULTURE: MECHANISMS OF CARDIOVASCULAR PROTECTION

1. The use of traditional Chinese medicinal herbs or their pharmaceutical products for disease prevention and management is becoming increasingly popular in Western countries. Mixtures of various Chinese herbs have been used for the treatment of syndromes clinically overlapping Western cardiovascular syndromes. One modern preparation, known as the ‘Cardiotonic Pill’ (CP), is a pharmaceutical product derived mainly from a medicinal herb, Salvia miltiorrhiza bunge, and recently widely used in Chinese hospitals for the prevention and management of ischaemic cardiovascular diseases. Although the CP is believed to confer an extensive range of benefits, little is known about the physiological actions of this medicine, particularly at the cellular and molecular levels. Therefore, the aim of the present study was to explore possible cellular mechanisms of the CP on the cardiovascular system.

Endogenous Estrogen Deficiency Reduces Proliferation and Enhances Apoptosis-Related Death in Vascular Smooth Muscle Cells Insights From the Aromatase-Knockout Mouse

Background—Altered proliferation and death of vascular smooth muscle cells (VSMCs) are important mechanisms in vascular growth and remodeling. This study examined the effect of endogenous estrogens on VSMC proliferation. Methods and Results—An estrogen-deficient animal model, the aromatase-knockout (ArKO) mouse, was used. Primary cultures of VSMCs were established from aortas of 11-week-old male and female ArKO and wild-type (WT) mice. In ArKO cells, the absence of aromatase cytochrome P450 mRNA expression was demonstrated by reverse transcription–polymerase chain reaction; Western blotting showed normal expression of estrogen receptor protein. Proliferative responses to serum or platelet-derived growth factor-BB were lower in ArKO than WT cells; 17&bgr;-estradiol (E2, 10 nmol/L) enhanced the response to platelet-derived growth factor-BB in ArKO cells but inhibited the response in WT cells. E2 inhibited activity of mitogen-activated protein kinase ERK1/2 in WT but not ArKO cells. Apoptosis-related death caused by tumor necrosis factor-&agr; was greater in ArKO than in WT cells; this effect in ArKO was attenuated by E2. Differences in VSMC proliferation between ArKO and WT occurred in both sexes. Morphological studies in aortas derived from male mice at 1 year of age demonstrated that medial smooth muscle area was ≈10% less in ArKO than WT mice at this age. Conclusions—Deficiency of endogenous estrogens reduces proliferation and enhances apoptosis-related death in VSMCs; exogenous E2 corrects these abnormalities.

Cellular mechanisms underlying the cardiovascular actions of oestrogens

Although pre-menopausal women enjoy relative cardiovascular protection, hormone (oestrogen+/-progestin)-replacement therapy has not shown cardiovascular benefits in post-menopausal women, suggesting that the effects of oestrogens on the cardiovascular system are much more complex than previously expected. Endothelial cells, smooth muscle cells, cardiac myocytes and fibroblasts, the cellular components of blood vessels and the heart, play important roles in cardiovascular health and disease. During the development and progression of cardiovascular disease, changes occur both in the structure and function of these cells, resulting in a wide range of abnormalities, which affect growth, death and physiological function. These cells contain functional oestrogen receptors and are targets for oestrogen action. This review focuses on recent studies on the effects of oestrogen on cardiovascular cell function. Oestrogens, particularly 17beta-oestradiol, exert multiple effects on cardiovascular cells, and these effects may contribute to the gender-associated protection against cardiovascular diseases.

Cell Adhesion Molecules as Pharmaceutical Target in Atherosclerosis

Cell adhesion molecules (CAMs) are transmembrane proteins that mediate adhesion and interactions between cells or cell and extra-cellular matrix. Increased expression and activation of CAMs in vascular endothelial cells and circulating leukocytes, as occurring in the settings of inflammation, hypercholesterolemia, hypertension and diabetes, stimulates leukocyte recruitment into the vascular endothelium, an important step in the pathogenesis of atherosclerosis. CAMs are a potential therapeutic target in clinical practice and in recent years pharmaceutical agents with specific effects on the production and function of these molecules have been studied and developed. This article reviews recent progress regarding pathophysiology of CAMs in atherogenesis and pharmaceutical products or chemicals that are active against CAMs, and assesses the possibilities for clinical developments in this area that might enhance the prevention, monitoring and treatment of atherosclerotic cardiovascular diseases.

Matrix-Dependent Gene Expression of Egr-1 and PDGF A Regulate Angiotensin II–Induced Proliferation in Human Vascular Smooth Muscle Cells

We have previously shown, in a neonatal rat cell line, that angiotensin II (Ang II)-induced proliferation in vascular smooth muscle cells is extracellular matrix (ECM) dependent. We hypothesized that such an effect might be mediated via differences in Ang II-induced increases in the transcriptional factor early growth response-1 (Egr-1) gene and, consequently, in platelet-derived growth factor (PDGF). Cultured human newborn aortic smooth muscle cells were studied on 4 different surfaces: (1) plastic, (2) laminin, (3) collagen, and (4) fibronectin. Ang II-induced increases in DNA synthesis were significantly greater on collagen (2.0+/-0.3-fold) and fibronectin (1.9+/-0.3-fold) than on laminin (1.0+/-0.2-fold) or plastic (1.4+/-0.2-fold). As with DNA synthesis, at 48 and 72 hours, Ang II-induced increases in cell numbers occurred only in cells grown on collagen and fibronectin culture plates and were blocked by an antagonist to the angiotensin type 1 (losartan, 10 micromol/L) but not the angiotensin type 2 (PD 123319, 10 micromol/L) receptor. Anti-PDGF AA antibody (6 microg/mL) blocked the increase in DNA synthesis by 60% to 64% in cells on collagen or fibronectin cultures but not on plastic cultures. When PDGF-AA (10 ng/mL) and Ang II were added together, DNA synthesis increased 2-fold and did not differ on the various ECM proteins. Increases in PDGF A-chain mRNA were observed only in cells grown on collagen (3.21+/-0.65-fold) and fibronectin (2.86+/-0.49-fold) plates 2 to 8 hours after the addition of Ang II and were blocked by losartan but not PD 123319. Expression of Egr-1, an early growth response gene, increased at 15 minutes, peaked at 30 minutes, and returned to normal after 2 hours with Ang II treatment. Ang II-induced increases in Egr-1 mRNA were greater on collagen (4. 82+/-0.66-fold at maximum) and fibronectin (4.01+/-0.56-fold) than on laminin (2.74+/-0.45-fold) or plastic (2.53+/-0.40-fold) and were blocked by losartan but not PD 123319. Thus, in human vascular smooth muscle cells in culture, Ang II-induced proliferation is mediated via the angiotensin type 1 receptor, dependent on ECM proteins, and regulated by differential gene expression of Egr-1 and PDGF-1.

A preparation of herbal medicine Salvia miltiorrhiza reduces expression of intercellular adhesion molecule-1 and development of atherosclerosis in apolipoprotein E-deficient mice.

Cardiotonic pill (CP) is a pharmaceutical preparation of the herbal medicine Salvia miltiorrhiza. In vitro studies demonstrate that CP inhibits vascular endothelial expression of adhesion molecules and smooth-muscle proliferation, implying the possibility of antiatherosclerotic effects. This study employs an in vivo animal model to examine the potential therapeutic efficacy of CP on atherosclerotic development. Male apolipoprotein E-deficient (ApoE−/−) mice fed with an atherogenic (high fat) diet were administered with CP (90-120 mg/kg per day) via drinking water for 8 weeks. Hypercholesterolemia developed in the mice, with 22-fold increases in plasma levels of total cholesterol, 29-fold of LDL, and 7-fold of HDL. CP therapy did not significantly alter the lipid levels. Expression of intercellular adhesion molecule 1 significantly increased in circulating leukocytes and was abolished by CP therapy. Atherosclerosis significantly developed in the aorta and was attenuated by CP therapy, with an approximately 30% reduction in whole atherosclerotic lesions and an approximately 50% reduction in fibrous plaques in the artery. Thus, herbal medicine CP partly protects ApoE−/− mice from high-fat diet-induced atherogenesis. The protection is unlikely to be attributable to decreases in circulating cholesterol levels, but it might possibly relate to an inhibition of expression of adhesion molecules and other effects that remain unknown at this time.

Estrogen inhibits mechanical strain-induced mitogenesis in human vascular smooth muscle cells via down-regulation of Sp-1

OBJECTIVE The cellular basis of the cardioprotective effects of estrogen are largely unknown. An inhibitory effect on vascular smooth muscle (VSM) growth has been proposed. We examined the effect of 17beta-estradiol (E2) on mechanical strain-induced mitogenesis in human fetal VSM cells. METHODS AND RESULTS Cells were grown on fibronectin-coated plates with silicone-elastomer bottoms, and exposed to cyclic mechanical strain (60 cycles/min), with and without E2 (1 nmol/l), for 48 h. [3H]-Thymidine incorporation was measured during the last 6 h. Strain induced 1.5-2 fold increases in DNA synthesis that were attenuated by antibodies to platelet-derived growth factor (PDGF) AA and BB. Strain also induced increases both in mRNA and protein levels of Sp-1, a transcription factor that binds to the PDGF-A gene promoter site. E2 attenuated strain-induced mitogenesis, and also increases in mRNA and protein levels of Sp-1. The estrogen receptor (ER) antagonist ICI 182,780 (100 nmol/l) reversed the inhibitory effect of E2 on strain-induced increases in DNA synthesis and Sp-1 protein. RT-PCR analysis showed presence of both ER-alpha and -beta in these cells. CONCLUSIONS Estrogen inhibits strain-induced mitogenesis in human VSM cells via an ER mediated process involving down-regulation of the transcription factor Sp-1.

A pharmaceutical preparation of Salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin II-stimulated collagen synthesis in fibroblasts

Salvia miltiorrhiza is a medicinal herb commonly used in traditional Chinese medicine for the prevention and treatment of cardiovascular disease. This study investigated the effects of Cardiotonic Pill (CP), a pharmaceutical preparation of Salvia miltiorrhiza, on cardiac myocytes and fibroblasts with respect to the viability, proliferation, and collagen synthesis in these cells under various conditions. A cardiac myocyte line, H9c2, and primarily cultured fibroblasts from rat hearts were incubated with CP over a broad concentration range (50-800 microg/ml) under normal cultures, conditions of ischemia (serum-free culture), and stimulation by angiotensin II (AII, 100 nM), hydrogen peroxide (H(2)O(2), 50-200 microM), or tumor necrosis factor alpha (TNFalpha, 40 ng/ml) for 24-48 h. Cell growth, apoptosis, DNA and collagen synthesis, and expression of relevant genes were assessed via cell number study, morphological examination, Annexin-V staining, flow-cytometry, [(3)H]-thymidine or [(3)H]-proline incorporation assay, and Western blotting analysis. It was found that (1) at therapeutic (50 microg/ml) and double therapeutic (100 microg/ml) concentrations, CP did not significantly affect normal DNA synthesis and cell growth in these cardiac cells, while at higher (over 4-fold therapeutic) concentrations (200-800 microg/ml), CP decreased DNA synthesis and cell growth and increased cell death; (2) CP treatment (50 microg/ml) significantly inhibited TNFalpha-induced apoptosis in myocytes, with 12.3+/-1.46% cells being apoptosis in CP treatment group and 37.0+/-7.34% in the control (p<0.01), and simultaneously, expression of activated (phosphorylated) Akt protein was increased by about 2 folds in the CP-treated cells; and (3) in cultured fibroblasts, CP significantly reduced AII-induced collagen synthesis in a concentration-dependent manner (by approximately 50% and approximately 90% reduction of AII-induced collagen synthesis at 50 and 100 microg/ml, respectively). Thus, Salvia miltiorrhiza preparation CP is physiologically active on cardiac cells. The actions by CP to reduce apoptotic damage in myocytes and collagen synthesis in fibroblasts may help to preserve the heart function and reduce heart failure risk. The actions by CP to inhibit DNA synthesis and cell growth, which occurred at over therapeutic doses, may weaken the ability of heart repair. Further studies are needed to identify the chemical compounds in this herbal product that are responsible for these observed physiological effects.

Cardiovascular Physiology of Androgens and Androgen Testosterone Therapy in Postmenopausal Women

Women before menopause are at relatively lower risk of cardiovascular disease (CVD) compared with age-matched men and after menopause this gender advantage disappears. Androgen has been known to be an independent factor contributing to the higher male susceptibility to CVD, through adverse effects on lipids, blood pressure, and glucose metabolism. High androgen levels also contribute to CVD development in women with polycystic ovary syndrome as well as androgen abusing athletes and body builders. On the other hand, decline in androgen levels, as a result of ageing in men, is associated with hypertension, diabetes and atherosclerosis. Postmenopausal women, particularly those with oophorectomy are generally in low levels of sex hormones and androgen insufficiency is independently associated with the higher incidence of atherosclerosis in postmenopausal women. Androgen testosterone therapy (ATT) has been commonly used to improve well-being and libido in aging men with low androgen levels. The therapy has been demonstrated also to effectively reduce atherogenesis in these people. The use of ATT in postmenopausal women has increased in recent years and to date, however, the cardiovascular benefits of such therapy in these women remain uncertain. This review focuses on research regarding the impact of endogenous androgens and ATT on the cardiovascular physiology and CVD development in postmenopausal women.

The isoflavone metabolite cis-tetrahydrodaidzein inhibits ERK-1 activation and proliferation in human vascular smooth muscle cells.

Abstract: Phytoestrogens have recently been proposed as alternatives to estrogens for cardiovascular protection; however, the effect of their metabolites on vascular biology is unclear. We studied the effect of a red clover-derived isoflavone metabolite cis-tetrahydrodaidzein (cis-THD) on human vascular smooth muscle cell (VSMC) proliferation. Cis-THD significantly inhibited platelet-derived growth factor (PDGF) BB-induced DNA synthesis (10% at 1 nmol/L, 17% at 10, 100 nmol/L; 17β-estradiol: 27% inhibition at 1, 10 nmol/L, 33% at 100 nmol/L). Cis-THD reduced PDGF BB-induced increase in cell numbers. Cis-THD showed high binding affinity to estrogen receptors (ER) by ER competitor assays; its inhibitory effect on DNA synthesis was abolished by the ER antagonist ICI 182780 (100 nmol/L), indicating ER-mediation. Immunoprecipitation assays revealed that cis-THD inhibited PDGF BB-stimulated activation of mitogen-activated protein (MAP) kinase ERK-1 by 34% at 1 nmol/L, 58% at 10 nmol/L, and 81% at 100 nmol/L, while MAP kinase JNK and p38 activities were unaltered. Thus, the isoflavone metabolite cis-THD inhibits PDGF-induced ERK-1 activation and cell proliferation in human VSMC, suggesting a potential beneficial effect in cardiovascular protection.