Donald M. Foster

Integrins and proximal signaling mechanisms in cardiovascular disease

Integrins are heterodimeric cell-surface molecules, which act as the principle mediators of molecular dialog between a cell and its extracellular matrix environment. In addition to their structural functions, integrins mediate signaling from the extracellular space into the cell through integrin-associated signaling and adaptor molecules such as FAK (focal adhesion kinase), ILK (integrin-linked kinase), PINCH (particularly interesting new cysteine-histidine rich protein) and Nck2 (non-catalytic (region of) tyrosine kinase adaptor protein-2). Via these molecules, integrin signaling tightly and cooperatively interacts with receptor tyrosine kinases (RTKs) signaling to regulate survival, proliferation and cell shape as well as polarity, adhesion, migration and differentiation. In the heart and blood vessels, the function and regulation of these molecules can be partially disturbed and thus contribute to cardiovascular diseases such as cardiac hypertrophy and atherosclerosis. In this review, we discuss the primary mechanisms of action and signaling of integrins in the cardiac and vascular system in normal and pathological states, as well as therapeutic strategies for targeting these systems (1).

Rac1 and RhoA differentially regulate angiotensinogen gene expression in stretched cardiac fibroblasts

AIMS Angiotensin II (Ang II) stimulates cardiac remodelling and fibrosis in the mechanically overloaded myocardium. Although Rho GTPases regulate several cellular processes, including myocardial remodelling, involvement in mediating mechanical stretch-induced regulation of angiotensinogen (Ao), the precursor to Ang II, remains to be determined. We, therefore, examined the role and associated signalling mechanisms of Rho GTPases (Rac1 and RhoA) in regulation of Ao gene expression in a stretch model of neonatal rat cardiac fibroblasts (CFs). METHODS AND RESULTS CFs were plated on deformable stretch membranes. Equiaxial mechanical stretch caused significant activation of both Rac1 and RhoA within 2-5 min. Rac1 activity returned to control levels after 4 h, whereas RhoA remained at a high level of activity until the end of the stretch period (24 h). Mechanical stretch initially caused a moderate decrease in Ao gene expression, but was significantly increased at 8-24 h. RhoA had a major role in mediating both the stretch-induced inhibition of Ao at 4 h and the subsequent upregulation of Ao expression at 24 h. β₁ integrin receptor blockade by Tac β₁ expression impaired acute (2 and 15 min) stretch-induced Rac1 activation, but increased RhoA activity. Molecular experiments revealed that Ao gene expression was inhibited by Rac1 through both JNK-dependent and independent mechanisms, and stimulated by RhoA through a p38-dependent mechanism. CONCLUSION These results indicate that stretch-induced activation of Rac1 and RhoA differentially regulates Ao gene expression by modulating p38 and JNK activation.

Stretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: Opposing roles of JNK1/2 and p38α MAP kinases

The cardiac renin-angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy, remodeling, and fibroblast proliferation in the hemodynamically overloaded heart. However, the intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a substrate of the RAS system, are largely unknown. Here we report the identification of JNK1/2 as a negative, and p38alpha as a major positive regulator of Ao gene expression. Isolated neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB) plated on deformable membranes coated with collagen IV, were exposed to 20% equiaxial static-stretch (0-24 h). Mechanical stretch initially depressed Ao gene expression (4 h), whereas after 8 h, Ao gene expression increased in a time-dependent manner. Blockade of JNK1/2 with SP600125 increased basal Ao gene expression in NRVM (10.52+/-1.98 fold, P<0.001) and NRFB (13.32+/-2.07 fold, P<0.001). Adenovirus-mediated expression of wild-type JNK1 significantly inhibited, whereas expression of dominant-negative JNK1 and JNK2 increased basal and stretch-mediated (24 h) Ao gene expression, showing both JNK1 and JNK2 to be negative regulators of Ao gene expression in NRVM and NRFB. Blockade of p38alpha/beta by SB202190 or p38alpha by SB203580 significantly inhibited stretch-induced (24 h) Ao gene expression, whereas expression of wild-type p38alpha increased stretch-induced Ao gene expression in both NRVM (8.41+/-1.50 fold, P<0.001) and NRFB (3.39+/-0.74 fold, P<0.001). Conversely, expression of dominant-negative p38alpha significantly inhibited stretch response. Moreover, expression of constitutively active MKK6b (E) significantly stimulated Ao gene expression in the absence of stretch, indicating that p38 activation alone is sufficient to induce Ao gene expression. Taken together p38alpha was demonstrated to be a positive regulator, whereas JNK1/2 was found to be a negative regulator of Ao gene expression. Prolonged stretch diminished JNK1/2 activation, which was accompanied by a reciprocal increase in p38 activation and Ao gene expression. This suggests that a balance in JNK1/2 and p38alpha activation determines the level of Ao gene expression in myocardial cells.

Integrins: Novel Therapeutic Targets for Cardiovascular Diseases

Integrins are the principle mediators of molecular dialog between a cell and its extracellular matrix environment. The unique combinations of integrin subunits determine which extracellular matrix molecules are recognized by a cell. Recent studies have demonstrated that remodeling in heart and vasculature is linked to alterations in extracellular matrix and integrin expression. The roles of integrins in controlling cellular behavior have made these molecules highly attractive drug targets. New insights into mechanisms whereby the extracellular matrix takes part in the control of smooth muscle cell proliferation and cardiac growth suggest a number of putative targets for future therapies that can be applied to increase plaque stability, prevent the clinical consequences of atherosclerosis and improve outcomes after interventional procedures such as cardiac transplantation. Therapeutic candidates include antibodies, cyclic peptides, peptidomimetics and small molecules. The integrin inhibitors Integrilin and ReoPro have been approved as blood thinners in cardiovascular disease, and newer agents are undergoing testing. Although integrin function is important in the cardiovascular system, there are wide gaps in knowledge. In this review, we discuss the primary mechanisms of action and signaling of integrins in the cardiac and vascular system in normal and pathological states, as well as therapeutic strategies for targeting these molecules in the cardiovascular system.

Characteristics of prostatc infarcts and their effect on serum prostate-specific antigen and prostatc acid phosphatase

Abstract Objectives. To determine how prostatic infarcts affect serum prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) levels. Methods. Two hundred eighteen clinically benign, whole prostates were obtained atautopsy, completely sectioned, and examined histologically. PSA and PAP levels were determined from premortem serum. Results. Six of the 218 (2.8%) prostates had infarcts. The infarcts were usually multipleand usually located in the central and/or middle concentric zones of the middle third of the prostate without a preference for a particular lobe. Serum PSA by immunoradiometric assay were elevated in all 6 cases. Serum PAP by both enzymatic assay (ACA), and immunoradiometric assay were available for 5 cases and were elevated by both methods in 2 cases, approached elevated levels by both methods in 1 case, and were normal by both methods in 2 cases. The PSA and PAP levels appeared to be affected more by the age than by the size of the infarct. Conclusions. Prostatic infarcts elevate PSA levels more frequently than PAP levels, andprostatic infarcts may be responsible for some unexplained elevations of serum PSA and PAP levels.

Effects of chronic alcohol consumption in weanling rats on brain gangliosides

Chronic ingestion of ethyl alcohol in pre-weanling rats can decrease whole-brain levels of sialic acid (SA), an acidic sugar that serves as terminal groups on glycolipids (gangliosides) and glycoproteins. Because SA occurs in both classes of membrane-bound chemicals, the alcohol effect could be on either or both parent compounds. We examined the effects of alcohol on gangliosides by measuring levels of six specific ganglioside species in post-weanling rats that were fed liquid alcohol diet for 35 days. We found no major effect on any of the ganglioside species in the alcohol-fed rats compared with their pair-fed littermates. These data suggest that alcohol may have acute effects on membrane gangliosides, but during chronic exposure in more mature animals, the membrane may adapt and maintain near-normal ganglioside composition. Thus, gangliosides may reflect mechanisms of membrane tolerance; they could also be involved in mediating metabolic dependencies in neuronal membranes, a possibility that needs testing.

Ethanol may stimulate or inhibit (Na+ + K+)-ATPase, depending upon Na+ and K+ concentrations

The influence of varying the ratios of [Na+]/[K+] on the effects of alcohol (500 mg/dl) on brain (Na+ + K+)-ATPase, using a commercial porcine enzyme preparation, showed that, generally, activity was stimulated by ethanol when [Na+] less than [K+], but inhibited when [Na+] greater than [K+] (with sum kept constant at 150 mM). In addition, when [Na+]/[K+] was 15/90 mM, representative of normal intracellular levels, ethanol (500 mg/dl) stimulated the porcine enzyme, but inhibited it when [Na+]/[K+] was 144/6 mM, representative of normal extracellular levels. Similarly, in freshly prepared enzyme from highly purified rat brain synaptic membranes, ethanol (100, 300, and 450 mg/dl) stimulated when [Na+]/[K+] was 15/88 mM (representing intracellular levels), but inhibited when [Na+]/[K+] was 142/4 mM (extracellular levels).

Evaluation of age-specific normal ranges for prostate-specific antigen.

OBJECTIVES To compare the traditional normal range (TNR) of 0.0 to 4.0 ng/mL for serum prostate-specific antigen (PSA) to age-specific normal ranges (ASNRs). METHODS An autopsy series of completely sectioned, clinically benign prostates from 171 consecutive Caucasian men over the age of 40 years was selected. These patients were divided into those having no prostate cancer at autopsy, prostate cancer less than 1 cc in volume, and prostate cancer at least 1 cc in volume. The PSA values of each group were compared using both the TNR and the ASNR. RESULTS Twenty-three of 105 (21.9%) patients with no cancer had elevated PSA values by the TNR, whereas only 18 (17.1%) were elevated using the ASNR. Nine of 54 (16.7%) with cancer less than 1 cc were elevated using the TNR, and 7 of 54 (13.0%) using the ASNR. Of 12 patients with cancer at least 1 cc, all had elevated PSA levels using the TNR and 11 (91.7%) were elevated using the ASNR. All discrepancies between the TNR and ASNR occurred in the 60- to 79-year age range. CONCLUSIONS Use of ASNRs appears helpful in increasing the specificity of PSA by eliminating some elevated values in patients in their 60s and 70s.

Molecular Signaling Mechanisms of Myocardial Stretch: Implications for Heart Disease

With every heartbeat, myocardial cells are subjected to substantial mechanical stretch. Stretch is a potent stimulus for growth, differentiation, migration, remodeling and gene expression. Mechanical load is a major cause of cardiac hypertrophy. Since the initial observation of stretch-induced growth, our understanding of this complex field has been steadily growing, but remains incomplete. The mechanisms by which myocardial cells convert mechanical stimuli into biochemical signals that result in physiologic and pathological changes remain to be completely understood. Integrins, caveolae and focal adhesions have been shown to have important mechanosensing roles in cardiac myocytes. Downstream effectors activated by mechanosensors include guanine-nucleotide binding proteins (G-proteins), mitogen-activated protein (MAP) kinases, Janus-associated kinase/signal transducers and activators of transcription (JAK/Stat), protein kinase C (PKC) and protein kinase B/Akt pathways. Multiple levels of crosstalk exist between these pathways. Early studies have implicated most of these pathways in cardiac injury and growth response, however, more recent advancements in the development of kinase-specific inhibitors and genetically-engineered animal models have revealed significant new insights. Recent studies suggest that acute mechanical stretch activates protective pathways including c-jun N-terminal kinase (JNK) and Akt as a tolerance response, rather than injury-related signaling cascades such as p38 MAP kinase. However, chronic stretch/mechanical load creates an imbalance that favors the injury related pathway by an unknown mechanism in the myocardium. The following chapter provides an overview of the fundamental processes of stretch-activated mechano-signaling in myocardial cells, and recent advances in our understanding of this increasingly important field.

Production of Spontaneously Beating Neonatal Rat Heart Tissue for Calcium and Contractile Studies

Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FB) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Typically, these cell types are separated using Percoll density gradient procedures. Cells located between the Percoll bands (interband cells [IBCs]), which contain less mature NRVM and a variety of non-myocytes, including coronary vascular smooth muscle cells and endothelial cells (ECs), are routinely discarded. However, we have demonstrated that IBCs readily attach to extracellular matrix-coated coverslips, plastic culture dishes, and deformable membranes to form a 2-dimensional cardiac tissue layer which quickly develops spontaneous contraction within 24 h, providing a robust coculture model for the study of cell-to-cell signaling and contractile studies. Below, we describe methods that provide good cell yield and viability of IBCs during isolation of NRVM and FB obtained from 0- to 3-day-old neonatal rat pups. Basic characterization of IBCs and methods for use in intracellular calcium and contractile experiments are also presented. This method maximizes the use of cells obtained from neonatal rat hearts.