Hemodynamic effects of epidermal growth factor in conscious rats and monkeys.

The therapeutic application of growth factors to human disease has become closer to reality with the advent of faster means of synthesizing these molecules and novel drug delivery strategies. Epidermal growth factor (EGF) belongs to a large family of molecules with the ability to modulate growth. Purified extracts of EGF have been used clinically to modulate gastrointestinal secretion of hormones and accelerate healing. EGF is also reported to have both vascular smooth muscle contractile and relaxing activity Cardiovascular studies were performed with the bioactive 48-amino acid fragment of human EGF in rodents and primates to determine the effects of EGF on blood pressure and heart rate in conscious animals. Intravenous infusion of EGF induced an initial pressor response in rats followed by a prolonged decrease in blood pressure. In contrast, in monkeys, EGF had dose-related blood pressure-lowering effects only; significant hypotension was observed at doses ranging from 3 to 300 microg/kg i.v. Hypotension was associated with modest tachycardia in both species. To our knowledge, this is the first report of hemodynamic effects of EGF in primates, and it clearly documents that the mitogenic role of growth factors such as EGF is but one aspect of their physiology.

Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1

Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory–fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.

Restoration of β-adrenergic signaling in failing cardiac ventricular myocytes via adenoviral-mediated gene transfer

Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β-adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β-adrenergic signaling defects including down-regulation of myocardial β-adrenergic receptors (β-ARs), functional β-AR uncoupling, and an up-regulation of the β-AR kinase (βARK1). Adenoviral-mediated gene transfer of the human β2-AR or an inhibitor of βARK1 to these failing myocytes led to the restoration of β-AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of βARK1 activity in the heart.

Mechanical stretch induces vascular permeability factor in human mesangial cells: Mechanisms of signal transduction

Hemodynamic abnormalities have been implicated in the pathogenesis of the increased glomerular permeability to protein of diabetic and other glomerulopathies. Vascular permeability factor (VPF) is one of the most powerful promoters of vascular permeability. We studied the effect of stretch on VPF production by human mesangial cells and the intracellular signaling pathways involved. The application of mechanical stretch (elongation 10%) for 6 h induced a 2.4-fold increase over control in the VPF mRNA level (P < 0.05). There was a corresponding 3-fold increase in VPF protein level by 12 h (P < 0.001), returning to the baseline by 24 h. Stretch-induced VPF secretion was partially prevented both by the protein kinase C (PKC) inhibitor H7 (50 μM: 72% inhibition, P < 0.05) and by pretreatment with phorbol ester (phorbol-12-myristate-13 acetate 10−7 M: 77% inhibition, P < 0.05). A variety of protein tyrosine kinase (PTK) inhibitors, genistein (20 μg/ml), herbimycin A (3.4 μM), and a specific pp60src peptide inhibitor (21 μM) also significantly reduced, but did not entirely prevent, stretch-induced VPF protein secretion (respectively 63%, 80%, and 75% inhibition; P < 0.05 for all). The combination of both PKC and PTK inhibition completely abolished the VPF response to mechanical stretch (100% inhibition, P < 0.05). Stretch induces VPF gene expression and protein secretion in human mesangial cells via PKC- and PTK-dependent mechanisms.

Vascular imprints of neuronal activity: Relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation

Modern functional neuroimaging methods, such as positron-emission tomography (PET), optical imaging of intrinsic signals, and functional MRI (fMRI) utilize activity-dependent hemodynamic changes to obtain indirect maps of the evoked electrical activity in the brain. Whereas PET and flow-sensitive MRI map cerebral blood flow (CBF) changes, optical imaging and blood oxygenation level-dependent MRI map areas with changes in the concentration of deoxygenated hemoglobin (HbR). However, the relationship between CBF and HbR during functional activation has never been tested experimentally. Therefore, we investigated this relationship by using imaging spectroscopy and laser-Doppler flowmetry techniques, simultaneously, in the visual cortex of anesthetized cats during sensory stimulation. We found that the earliest microcirculatory change was indeed an increase in HbR, whereas the CBF increase lagged by more than a second after the increase in HbR. The increased HbR was accompanied by a simultaneous increase in total hemoglobin concentration (Hbt), presumably reflecting an early blood volume increase. We found that the CBF changes lagged after Hbt changes by 1 to 2 sec throughout the response. These results support the notion of active neurovascular regulation of blood volume in the capillary bed and the existence of a delayed, passive process of capillary filling.

Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction

Previous studies have shown that proinflammatory cytokines, such as tumor necrosis factor (TNF), are expressed after acute hemodynamic overloading and myocardial ischemia/infarction. To define the role of TNF in the setting of ischemia/infarction, we performed a series of acute coronary artery occlusions in mice lacking one or both TNF receptors. Left ventricular infarct size was assessed at 24 h after acute coronary occlusion by triphenyltetrazolium chloride (TTC) staining in wild-type (both TNF receptors present) and mice lacking either the type 1 (TNFR1), type 2 (TNFR2), or both TNF receptors (TNFR1/TNFR2). Left ventricular infarct size as assessed by TTC staining was significantly greater (P < 0.005) in the TNFR1/TNFR2-deficient mice (77.2% ± 15.3%) when compared with either wild-type mice (46.8% ± 19.4%) or TNFR1-deficient (47.9% ± 10.6%) or TNFR2-deficient (41.6% ± 16.5%) mice. Examination of the extent of necrosis in wild-type and TNFR1/TNFR2-deficient mice by anti-myosin Ab staining demonstrated no significant difference between groups; however, the peak frequency and extent of apoptosis were accelerated in the TNFR1/TNFR2-deficient mice when compared with the wild-type mice. The increase in apoptosis in the TNFR1/TNFR2-deficient mice did not appear to be secondary to a selective up-regulation of the Fas ligand/receptor system in these mice. These data suggest that TNF signaling gives rise to one or more cytoprotective signals that prevent and/or delay the development of cardiac myocyte apoptosis after acute ischemic injury.

Lack of agreement between tonometric and gastric juice partial carbon dioxide tension

Our goal was to compare measurement of tonometered saline and gastric juice partial carbon dioxide tension (PCO2). In this prospective observational study, 112 pairs of measurements were simultaneously obtained under various hemodynamic conditions, in 15 critical care patients. Linear regression analysis showed a significant correlation between the two methods of measuring PCO2 (r 2 = 0.43; P < 0.0001). However, gastric juice PCO2 was systematically higher (mean difference 51 mmHg). The 95% limits of agreement were 315 mmHg and the dispersion increased as the values of PCO2 increased. Tonometric and gastric juice PCO2 cannot be used interchangeably. Gastric juice PCO2 measurement should be interpreted with caution.

Event-related functional MRI: Past, present, and future

The past two decades have seen an enormous growth in the field of human brain mapping. Investigators have extensively exploited techniques such as positron emission tomography and MRI to map patterns of brain activity based on changes in cerebral hemodynamics. However, until recently, most studies have investigated equilibrium changes in blood flow measured over time periods upward of 1 min. The advent of high-speed MRI methods, capable of imaging the entire brain with a temporal resolution of a few seconds, allows for brain mapping based on more transient aspects of the hemodynamic response. Today it is now possible to map changes in cerebrovascular parameters essentially in real time, conferring the ability to observe changes in brain state that occur over time periods of seconds. Furthermore, because robust hemodynamic alterations are detectable after neuronal stimuli lasting only a few tens of milliseconds, a new class of task paradigms designed to measure regional responses to single sensory or cognitive events can now be studied. Such “event related” functional MRI should provide for fundamentally new ways to interrogate brain function, and allow for the direct comparison and ultimately integration of data acquired by using more traditional behavioral and electrophysiological methods.

Imaging the functioning human brain

One of the most exciting methodological advances for brain research field arises in functional brain imaging, which enables us to localize and characterize neural activity and biochemical events in the living human brain. Recently developed event-related functional MRI makes it possible to visualize the brain activity associated with cognitive processes with the temporal resolution of the hemodynamic response. In addition, the high sensitivity and selectivity of positron-emission tomography allow us to probe the neurochemical processes at the molecular level. Positron-emission tomography also has been applied to investigate the effects of therapeutic drugs as well as the effects of drugs of abuse.

Up-regulation of pressure-activated Ca(2+)-permeable cation channel in intact vascular endothelium of hypertensive rats.

In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2(+)-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.

Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel.

At birth, pulmonary vasodilation occurs as air-breathing life begins. The mechanism of O2-induced pulmonary vasodilation is unknown. We proposed that O2 causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel (KCa) via a cyclic nucleotide-dependent kinase. We tested this hypothesis in hemodynamic studies in acutely prepared fetal lambs and in patch-clamp studies on resistance fetal pulmonary artery smooth muscle cells. Fetal O2 tension (PaO2) was increased by ventilating the ewe with 100% O2, causing fetal total pulmonary resistance to decrease from 1.18 +/- 0.14 to 0.41 +/- 0.03 mmHg per ml per min. Tetraethylammonium and iberiotoxin, preferential KCa-channel inhibitors, attenuated O2-induced fetal pulmonary vasodilation, while glibenclamide, an ATP-sensitive K+-channel antagonist, had no effect. Treatment with either a guanylate cyclase antagonist (LY83583) or cyclic nucleotide-dependent kinase inhibitors (H-89 and KT 5823) significantly attenuated O2-induced fetal pulmonary vasodilation. Under hypoxic conditions (PaO2 = 25 mmHg), whole-cell K+-channel currents (Ik) were small and were inhibited by 1 mM tetraethylammonium or 100 nM charybdotoxin (CTX; a specific KCa-channel blocker). Normoxia (PaO2 = 120 mmHg) increased Ik by more than 300%, and this was reversed by 100 nM CTX. Nitric oxide also increased Ik. Resting membrane potential was -37.2 +/- 1.9 mV and cells depolarized on exposure to CTX, while hyperpolarizing in normoxia. We conclude that O2 causes fetal pulmonary vasodilation by stimulating a cyclic nucleotide-dependent kinase, resulting in KCa-channel activation, membrane hyperpolarization, and vasodilation.

Arrest of endotoxin-induced hypotension by transforming growth factor beta1.

Septic shock is a cytokine-mediated process typically caused by a severe underlying infection. Toxins generated by the infecting organism trigger a cascade of events leading to hypotension, to multiple organ system failure, and frequently to death. Beyond supportive care, no effective therapy is available for the treatment of septic shock. Nitric oxide (NO) is a potent vasodilator generated late in the sepsis pathway leading to hypotension; therefore, NO represents a potential target for therapy. We have previously demonstrated that transforming growth factor (TGF) beta1 inhibits inducible NO synthase (iNOS) mRNA and NO production in vascular smooth muscle cells after its induction by cytokines critical in the sepsis cascade. Thus, we hypothesized that TGF-beta1 may inhibit iNOS gene expression in vivo and be beneficial in the treatment of septic shock. In a conscious rat model of septic shock produced by Salmonella typhosa lipopolysaccharide (LPS), TGF-beta1 markedly reduced iNOS mRNA and protein levels in several organs. In contrast, TGF-beta1 did not decrease endothelium-derived constitutive NOS mRNA in organs of rats receiving LPS. We also performed studies in anesthetized rats to evaluate the effect of TGF-beta1 on the hemodynamic compromise of septic shock; after an initial 25% decrease in mean arterial pressure, TGF-beta1 arrested LPS-induced hypotension and decreased mortality. A decrease in iNOS mRNA and protein levels in vascular smooth muscle cells was demonstrated by in situ hybridization and NADPH diaphorase staining in rats treated with TGF-beta1. Thus these studies suggest that TGF-beta1 inhibits iNOS in vivo and that TGF-beta1 may be of future benefit in the therapy of septic shock.

Telomere length and replicative aging in human vascular tissues.

Because repeated injury of the endothelium and subsequent turnover of intimal and medial cells have been implicated in atherosclerosis, we examined telomere length, a marker of somatic cell turnover, in cells from these tissues. Telomere lengths were assessed by Southern analysis of terminal restriction fragments (TRFs) generated by HinfI/Rsa I digestion of human genomic DNA. Mean TRF length decreased as a function of population doublings in human endothelial cell cultures from umbilical veins, iliac arteries, and iliac veins. When endothelial cells were examined for mean TRF length as a function of donor age, there was a significantly greater rate of decrease for cells from iliac arteries than from iliac veins (102 bp/yr vs. 47 bp/yr, respectively, P < 0.05), consistent with higher hemodynamic stress and increased cell turnover in arteries. Moreover, the rate of telomere loss as a function of donor age was greater in the intimal DNA of iliac arteries compared to that of the internal thoracic arteries (147 bp/yr vs. 87 bp/yr, respectively, P < 0.05), a region of the arterial tree subject to less hemodynamic stress. This indicates that the effect is not tissue specific. DNA from the medial tissue of the iliac and internal thoracic arteries showed no significant difference in the rates of decrease, suggesting that chronic stress leading to cellular senescence is more pronounced in the intima than in the media. These observations extend the use of telomere size as a marker for the replicative history of cells and are consistent with a role for focal replicative senescence in cardiovascular diseases.