The versatility and paradox of GDF 11.

In addition to its roles in embryonic development, Growth and Differentiation Factor 11 (GDF 11) has recently drawn much interest about its roles in other processes, such as aging. GDF 11 has been shown to play pivotal roles in the rescue of the proliferative and regenerative capabilities of skeletal muscle, neural stem cells and cardiomyocytes. We would be remiss not to point that some controversy exists regarding the role of GDF 11 in biological processes and whether it will serve as a therapeutic agent. The latest studies have shown that the level of circulating GDF 11 correlates with the outcomes of patients with cardiovascular diseases, cancer and uremia. Based on these studies, GDF 11 is a promising candidate to serve as a novel biomarker of diseases. This brief review gives a detailed and concise view of the regulation and functions of GDF 11 and its roles in development, neurogenesis and erythropoiesis as well as the prospect of using this protein as an indicator of cardiac health and aging.

Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy.

Tumor vessels are characterized by abnormal morphology and hyperpermeability that together cause inefficient delivery of chemotherapeutic agents. Although vascular endothelial growth factor has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here we show that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TECs exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards extracellular matrix stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 knockout mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anticancer drug cisplatin, significantly reduced tumor growth in wild-type mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

Regulation of shear stress in the canine coronary microcirculation.

BACKGROUND: Physical forces, such as pressure and flow, are well known to affect vascular function in the coronary circulation. Increases in shear stress produce vasodilation in coronary arterioles in vitro, and constant-flow preparations suggest a role for shear stress-induced vasodilation during adjustments to metabolic demand in vivo. Hypothetically, the regulation of shear stress can be viewed as a negative feedback control scheme (increased velocity --> increased shear --> vasodilation --> decreased velocity --> shear normalized). Therefore, we hypothesized that shear stress would be at least partially regulated during conditions of elevated flow. METHODS AND RESULTS: We used fluorescence microangiography to measure microvascular diameters and velocities in the coronary circulation in vivo and used these variables to calculate shear stress. Measurements were obtained under basal conditions, during maximal coronary blood flow, and after inhibition of NO synthase. Basal shear stress in the coronary circulation averaged 10 dyn/cm2 in small arteries and 19 dyn/cm2 in arterioles. Regulation of shear stress was observed in small arteries during adenosine-induced increases in coronary blood flow, but arterioles showed minimal regulation. NO synthase blockade had no effect on basal shear stress but completely abolished its regulation in small arteries during vasodilation. CONCLUSIONS: Our data provide the first quantitative estimates of microvascular shear stress in the coronary circulation. Moreover, our results suggest that shear stress in small coronary arteries is regulated by NO release from the endothelium.

Ischemia-induced coronary collateral growth is dependent on vascular endothelial growth factor and nitric oxide.

BACKGROUND: We hypothesized that ischemia-induced expression of vascular endothelial growth factor (VEGF) and the production of NO stimulate coronary collateral growth. METHODS AND RESULTS: To test this hypothesis, we measured coronary collateral blood flow and VEGF expression in myocardial interstitial fluid in a canine model of repetitive myocardial ischemia under control conditions and during antagonism of NO synthase. Collateralization was induced by multiple (1/h; 8/d), brief (2 minutes) occlusions of the left anterior descending coronary artery for 21 days. In controls, collateral blood flow (microspheres) progressively increased to 89+/-9 mL. min(-1). 100 g(-1) on day 21, which was equivalent to perfusion in the normal zone. Reactive hyperemic responses (a measure of the severity of ischemia) decreased as collateral blood flow increased. In N(G)-nitro-L-arginine methyl ester (L-NAME)- and L-NAME+nifedipine-treated dogs, to block the production of NO and control hypertension, respectively, collateral blood flow did not increase and reactive hyperemia was robust throughout the occlusion protocol (P<0.01 versus control). VEGF expression (Western analyses of VEGF(164) in myocardial interstitial fluid) in controls peaked at day 3 of the repetitive occlusions but waned thereafter. In sham-operated dogs (instrumentation but no occlusions), expression of VEGF was low during the entire protocol. In contrast, VEGF expression was elevated throughout the 21 days of repetitive occlusions after L-NAME. Reverse transcriptase-polymerase chain reaction analyses revealed that the predominant splice variant expressed was VEGF(164). CONCLUSIONS: NO is an important regulator of coronary collateral growth, and the expression of VEGF is induced by ischemia. Furthermore, the induction of coronary collateralization by VEGF appears to require the production of NO.

Basic fibroblast growth factor and heparin influence coronary arteriolar tone by causing endothelium-dependent dilation.

OBJECTIVE: The strong angiogenic and mitogenic agents acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) share signalling pathways with known vasodilatory agonists. Therefore, we hypothesized the FGF's produce vasoactive responses. We also proposed that heparin would exert a similar action to FGF, because this proteoglycan not only binds to the FGF receptor, but also facilitates the release of FGF from the cardiac extracellular matrix and promotes its binding to a high-affinity receptor. To test these hypotheses, we examined the vasodilatory reactions of coronary arterioles to aFGF, bFGF, and heparin, and the effects of antagonists to nitric oxide synthase (L-NMMA), prostaglandins (indomethacin), ATP-sensitive potassium (K[ATP]) channels (glibenclamide), FGF and FGF receptors on the vasoactive responses. METHODS: Arterioles (70-110 microns, internal diameter) were dissected from pig hearts and cannulated with micropipettes. Diameter was determined with videomicroscopy in response to bFGF and aFGF in concentrations of 1-100 ng/ml and to heparin (5-200 U/ml). RESULTS: Basic FGF, but not aFGF, caused dose-dependent vasodilation with a maximum of 61 +/- 4%. Relaxation of bFGF was antagonized by pretreatment with L-NMMA, but was not affected by pretreatment with indomethacin or glibenclamide. Heparin caused dose-dependent vasodilation with a maximum of 100 +/- 3% which was partially blocked by either L-NMMA or glibenclamide, but not by indomethacin. Furthermore, the effect of bFGF could be significantly blocked by pretreatment with an FGF receptor antibody as well as with a monoclonal antibody against FGF. Pretreatment with both antibodies significantly inhibited also the effect of heparin. CONCLUSIONS: These results indicate that bFGF and heparin cause vasodilation of coronary arterioles via an increase in NO production and heparin additionally by other mechanisms such as by activating K[ATP] channels. Furthermore, the effect of heparin is partially mediated via FGF and FGF receptors. We therefore speculate that both substances may be involved in the regulation of coronary microvascular tone acting partially through the same signalling mechanisms.

Integrated regulation of pressure and flow in the coronary microcirculation.

There are many mechanisms that contribute to the regulation of coronary blood flow including vasodilatory metabolites, myogenic regulation, flow- or shear stress-mediated vasodilation, and neurohumoral influences. It is interesting to note that coronary arterioles of varying sizes appear to possess different sensitivities to these regulatory factors, but each appears to dominate the control of a particular segment of the microvasculature. For example, during metabolic hyperemia the smallest arterioles seem to be most sensitive to the effects of metabolites, but metabolic and myogenic mechanisms that dictate the tone of upstream microvessels likely act in concert to facilitate the overall adjustments in coronary vasomotor tone. Neurohumoral mechanisms, seem to modulate the robustness of these intrinisic adjustments, perhaps by restraining the extent of vasodilation. The purpose of this review is to discuss these many regulatory mechanisms and also present a framework by which the vasoactive reactions elicited by these different mechanisms are integrated into coordinated responses of the entire coronary vascular network.

Multifactorial basis for coronary collateralization: a complex adaptive response to ischemia.

Angiogenesis and vasculogenesis are adaptive responses of the coronary collateral circulation to myocardial ischemia. This review focuses on the concerted action of growth factors, growth factor receptors, extracellular matrix, and inflammatory cellular responses to regulate angiogenesis and vasculogenesis in response to myocardial ischemia and alterations in shear stress. Therapeutic angiogenesis represents a novel approach to increase myocardial perfusion in patients with coronary artery disease and provides an opportunity to further clarify the mechanisms that regulate collateral development. Impairment of angiogenic adaptive responses to ischemia during disease states is an important subject for future investigation.

Actin isoform and alpha 1B-adrenoceptor gene expression in aortic and coronary smooth muscle is influenced by cyclical stretch.

The occurrence of vascular domains with specific biological and pharmacological characteristics suggests that smooth muscle cells in different arteries may respond differentially to a wide range of environmental stimuli. To determine if some of these vessel-specific differences may be attributable to mechano-sensitive gene regulation, the influence of cyclical stretch on the expression of actin isoform and alpha 1B-adrenoceptor genes was examined in aortic and coronary smooth muscle cells. Cells were seeded on an elastin substrate and subjected to maximal stretching (24% elongation) and relaxation cycles at a frequency of 120 cycles/min in a Flexercell strain unit for 72 h. Total RNA was extracted and hybridized to radiolabeled cDNA probes to assess gene expression. Stretch caused a greater reduction of actin isoform mRNA levels in aortic smooth muscle cells as compared to cells from the coronary artery. Steady-state mRNA levels of alpha 1B-adrenoceptor were also decreased by cyclical stretch in both cell types but the magnitude of the response was greater in coronary smooth muscle cells. No changes in alpha 1B-adrenoceptor or beta/gamma-actin steady-state mRNA levels were observed in H4IIE cells, a nonvascular, immortalized cell line. The relative gene expression of heat shock protein 70 was not influenced by the cyclic stretch regimen in any of these cell types. These results suggest that stretch may participate in the regulation of gene expression in vascular smooth muscle cells and that this response exhibits some degree of cell-specificity.

Distribution and control of coronary microvascular resistance.

Coronary blood flow depends upon the vascular resistance distributed non-uniformly within the coronary microcirculation. Coronary microvascular resistance is governed by metabolic, myogenic, endothelial and neurohumoral influences. A number of these control mechanisms interact locally within a microvascular segment. In addition, these control mechanisms occupy differing longitudinal response gradients, a feature which maximizes their potential for the synergistic control of flow.

Effects of transforming growth factor-beta and mechanical strain on osteoblast cell counts: an in vitro model for distraction osteogenesis.

Factors known to regulate bone production during distraction osteogenesis include mechanical strain on bone forming cells and up-regulation of transforming growth factor-beta (TGF-beta) during the distraction, or strain phase of distraction osteogenesis. In the present study, an in vitro model was used to evaluate the functional effect of exogenous TGF-beta1 on mitogenesis in murine-derived MC3T3 osteoblasts during the period of active mechanical strain. The first hypothesis to be tested was that mitogenic suppression of MC3T3 osteoblasts by TGF-beta1 is further enhanced when these cells are also subjected to mechanical strain. To test this hypothesis, MC3T3 osteoblasts were seeded on flexible and rigid membranes. These were subjected to cyclic, vacuum-induced strain, simulating physiologic stress loads. After 24 hours, all cells were transferred to media containing TGF-beta1, and strain was continued for an additional 48 hours. The study was repeated by using two doses of TGF-beta1. This study demonstrated that final cell counts were significantly decreased in the presence of TGF-beta1 in both the nonstrained and strained groups (p < 0.0001). The final cell count in the strained group was significantly less than that in the nonstrained group (p < 0.0001) for both concentrations of TGF-beta1 tested, confirming the initial hypothesis. The second hypothesis to be tested was that alteration in the mitogenic response of MC3T3 osteoblasts after strain is not directly due to autocrine factors produced by the strained osteoblasts. To test this hypothesis, a proliferation assay was performed on nonconfluent MC3T3 osteoblasts by using conditioned media collected from strained and nonstrained osteoblasts. This study demonstrated no significant differences in cell counts after addition of conditioned media collected from strained versus nonstrained cells, confirming the latter hypothesis. The present study demonstrates the functional significance of mechanical strain on osteoblast cell counts. Furthermore, this may help to explain the temporal relationship observed during the early distraction (strain) phase of distraction osteogenesis in rodent models in which peak up-regulation of TGF-beta1 gene expression correlates with peak suppression of osteoblast function as measured by gene expression of extracellular matrix proteins.

Coronary microcirculation in health and disease. Summary of an NHLBI workshop.

This article summarizes a 2-day workshop on the coronary microcirculation held in Bethesda, Md, in September 1994 and sponsored by the National Heart, Lung, and Blood Institute of the National Institutes of Health. The workshop explored a variety of topics pertaining to coronary microvascular physiology and pathophysiology. The latest methodologies that are being used to investigate the coronary microvasculature, including endoscopic microscopy of the intramural coronary microvasculature and micro-x-ray computerized tomography, were discussed. The most recent advances in the regulation of the coronary microcirculation-for example, myogenic and flow-dependent responses, KATP channels, and regional heterogeneity-were reported. The workshop touched on the relation of the microcirculation to clinically important conditions and offered recommendations for future research in this important area. Comparisons are made to recent advances in the peripheral circulation and current gaps in our knowledge concerning the coronary microcirculation. In recent years, research on the coronary microcirculation has made substantial advances, in part as a result of investigations in the peripheral microcirculation but also because of the application of unique methodologies. This research is providing new ways to investigate abnormalities of myocardial perfusion, an area of inquiry that until recently has been limited to examination of coronary pressure-flow relationships.

Microvascular pressures and resistances in the left ventricular subepicardium and subendocardium.

These experiments tested the hypothesis that differences in the distribution of subepicardial and subendocardial microvascular resistances may alter the transmural distributions of microvascular pressures. Isolated blood- and physiological saline-perfused porcine hearts were surgically incised to enable exposure of the subendocardial and subepicardial microcirculations. Microvascular pressures were measured during cardiac arrest and maximal vasodilation at various perfusion pressures to formulate relations between perfusion pressure and microvascular pressure in the different subendocardial (both free wall and papillary muscle) and subepicardial segments. Measurements of arteriolar and venular pressures in both myocardial regions were performed in comparably sized vessels (80-120 microns in diameter). At a coronary perfusion pressure of 100 mm Hg, subendocardial arteriolar and venular pressures were 60 +/- 4 and 33 +/- 3 mm Hg, respectively. In contrast, at the same coronary perfusion pressure, arteriolar and venular pressures in the subepicardial microcirculation averaged 80 +/- 6 and 22 +/- 3 mm Hg, respectively (p less than 0.05 versus subendocardium). At all levels of coronary perfusion pressure, arteriolar pressures were significantly lower in the subendocardium than in the subepicardium (p less than 0.05). Venular pressures were also higher in the subendocardial microcirculation than in the subepicardial microcirculation at all but the lowest perfusion pressure (p less than 0.05). The relative distribution of resistances in arteries, microvessels, and veins was also different between the subepicardium and subendocardium. Specifically, in the subendocardium, arterial and venous resistances were higher, percentage-wise, but microvascular resistance was proportionately lower than that in the subepicardium (p less than 0.05). From these data, it is concluded that the distribution of microvascular resistances and pressures is different during maximal vasodilation in the subepicardial and subendocardial microcirculations of the left ventricle. It is also speculated that differences in autoregulatory capacity and vulnerability to ischemia may be partially related to unequal distribution of microvascular resistances across the wall of the left ventricle.

Functional distribution of alpha 1- and alpha 2-adrenergic receptors in the coronary microcirculation.

BACKGROUND: The goal of this study was to determine the functional distribution of alpha 1- and alpha 2-adrenergic receptors in the epicardial coronary microcirculation. This goal was accomplished by intracoronary administration of the selective alpha 1-adrenergic agonist phenylephrine and the selective alpha 2-adrenergic agonist BHT-933 during measurements of coronary microvascular diameters in the beating heart. METHODS AND RESULTS: Experimental measurements were made under conditions with intact vasomotor tone and during coronary hypoperfusion (i.e., under conditions with autoregulatory mechanisms intact and blunted, respectively). Administration of selective alpha 1- and alpha 2-adrenergic antagonists, prazosin and SKF 104078, respectively, confirmed that the agonists were preferentially activating the desired adrenergic receptor subtype because the vasoconstrictor effects of the agonists were completely blocked by the appropriate antagonist. With baseline coronary vasomotor tone intact, phenylephrine caused constriction (8 +/- 3% decrease in diameter, p less than 0.05) of small coronary arteries (vessels greater than 100 microns in diameter) but did not produce constriction of coronary arterioles (vessels less than 100 microns in diameter). During coronary hypoperfusion, phenylephrine caused constriction (p less than 0.05) of both small coronary arteries and arterioles, 6 +/- 2% and 11 +/- 3% decreases in diameter, respectively. BHT-933 did not cause significant changes in microvascular diameters under control conditions but substantially and selectively decreased arteriolar diameters during hypoperfusion (24 +/- 6% decrease in diameter, p less than 0.05). CONCLUSIONS: In the intact, autoregulating coronary circulation, coronary arterioles escape from the effects of adrenergic activation but coronary arteries do not; rather, they can exhibit alpha 1-adrenergic coronary vasoconstriction. During coronary hypoperfusion, when autoregulatory adjustments are blunted, coronary arterioles are sensitive to both alpha 1- and alpha 2-adrenergic agonists, demonstrating significant constrictor responses. Also, the magnitude of coronary alpha 2-adrenergic arteriolar constriction (24% decrease in diameter) is significantly greater than that of alpha 2-adrenergic constriction (11% decrease in diameter) (p less than 0.05). Thus, alpha 1- and alpha 2-adrenergic activation produce different constrictor effects in the coronary microcirculation under baseline conditions when autoregulatory adjustments are intact and during coronary hypoperfusion when autoregulation is blunted. The data suggest that alpha 2-adrenergic receptors are preferentially distributed in arterioles, whereas alpha 1-adrenergic receptors are located throughout the coronary microcirculation. Importantly, the data also suggest that intrinsic autoregulatory adjustments in tone (i.e., autoregulatory escape) can override either alpha 1- or alpha 2-adrenergic constriction in coronary arterioles.

Adrenergic vasomotion in the coronary microcirculation.

The goal of this study was to determine the alpha-adrenergic receptor subtype(s) responsible for constriction at different microvascular levels in the coronary circulation. To accomplish these goals, the epicardial coronary microcirculation of intact beating hearts was viewed through an intravital microscope using stroboscopic epi-illumination. An initial study was designed to establish sites of alpha-adrenergic constriction to norepinephrine in preparations with intact vasomotor tone. For the primary experimental goal, coronary microvascular responses to selective alpha 1-adrenergic (phenylephrine) or alpha 2-adrenergic (BHT-933) agonists were evaluated, when coronary autoregulatory escape mechanisms were blunted during hypoperfusion. Infusion of norepinephrine decreased diameter of arterial vessels greater than 100 microns in diameter, but downstream coronary arterioles dilated significantly, representing autoregulatory escape from adrenergic vasoconstriction. In studies designed to examine the adrenergic receptor subtype (during hypoperfusion), phenylephrine produced modest constriction of vessels throughout the microcirculation (6-9% decrease in diameter), whereas BHT-933 produced marked constriction of small coronary microvessels, those less than 100 microns in diameter (24% decrease in diameter). From these results we conclude: 1) norepinephrine infusion causes disparate responses in the coronary microvasculature: constriction occurs in vessels greater than 100 microns in diameter, but dilation, via autoregulatory escape, predominates in vessels less than 100 microns in diameter; 2) alpha 1-adrenergic receptors are located in coronary arterioles and arteries; and 3) alpha 2-adrenergic receptors are preferentially located in small coronary arterioles. Thus, alpha 1- and alpha 2-adrenergic activation can produce dissimilar constrictor effects in the coronary microcirculation during hypoperfusion.