Endothelial dysfunction is present only in the microvasculature and microcirculation of early diffuse systemic sclerosis patients.

OBJECTIVES: To evaluate endothelial function and vascular stiffness in large, medium, small and microcirculatory blood vessels in very early diffuse systemic sclerosis (SSc). METHODS: We studied consecutive early diffuse SSc patients, defined as <2 years from first SSc symptom who did not have a prior cardiovascular event. Age, gender and race-matched controls were recruited. All underwent assessment of aortic pulse wave velocity (PWV), carotid intima-media thickness (IMT) brachial flow-mediated dilation (FMD), digital peripheral artery tonometer (EndoPAT) assessment and laser speckle contrast imaging (LSCI). RESULTS: Fifteen early diffuse SSc and controls were evaluated. The average age was 49 years, 63% were female and 93% were Caucasian. There were no differences in body mass index, hypertension, diabetes or hyperlipidaemia between controls and SSc patients. Mean SSc disease duration was 1.3 years. In the large central vessels, there was no difference in aortic PWV (p=0.71) or carotid IMT (p=0.92) between SSc patients and controls. Similarly, there was no difference in endothelial dysfunction with brachial artery FMD after ischaemia (p=0.55) and nitroglycerin administration (p=0.74). There were significantly lower values for digital EndoPAT measures (p=0.0001) in SSc patients. LSCI revealed a distinct pattern of microcirculatory abnormalities in response to ischaemia in SSc patients compared to controls. Imaging demonstrated a blunted microcirculatory hyperaemia of the hand with greater subsequent response to nitroglycerin. CONCLUSIONS: These findings suggest that the earliest endothelial changes occur in smaller arterioles and microvascular beds, but not in medium or macrovascular beds, in early diffuse SSc.

A Novel Acquisition Technique to Utilize Swan-Ganz Catheter data as a Surrogate for High-fidelity Micromanometry within the Right Ventricle and Pulmonary Circuit.

We explored the possibility of using conventional right-heart catheterization data, gathered both prospectively and retrospectively, as a surrogate for high-fidelity micro-manometery when analyzing systolic and diastolic RV function and calculating various ventricular and pulmonary hemodynamic parameters in the time domain. Right heart catheterizations were performed on 13 patients (7 female), who were suspected of having pulmonary hypertension. The procedure included use of both fluid-filled catheter and high-fidelity micromanometry to measure right ventricular and pulmonary arterial pressures. A digital data acquisition system was used to record micromanometer readings and data from the fluid-filled catheter system during prospective portion of the study. Retrospective data was obtained by direct digitization of screen captures taken by the conventional clinical system (fluid-filled catheter). From the 13 patients, 12-13 RV waveforms and 12 PA waveforms were acquired from each method. Basic measurements of heart rate, systolic pressure, diastolic pressure, dP/dtmax, and dP/dtmin were compared between micromanometry, direct acquisition from the PA catheter (voltage acquisition), and re-digitization of the hemodynamic waveforms (tracing). Correlation between Swan and tracing was stronger than that of Millar and Swan. SBP, followed by HR, has the strongest correlation of any parameter for all three methods, while DBP appears to be the weakest. Bland-Altman analysis shows all parameters to have minimal biases that are within clinical limits. Interoperator and intraoperator variability was minimal. Digital right-heart catheterization (RHC) data can be used as a surrogate for micromanometric data under ideal conditions for hemodynamic measures in the time domain. Pre-existing RHC data can be re-digitized for more rigorous hemodynamic analysis.

NADPH oxidase-derived ROS and the regulation of pulmonary vessel tone.

Pulmonary vessel constriction results from an imbalance between vasodilator and vasoconstrictor factors released by the endothelium including nitric oxide, endothelin, prostanoids, and reactive oxygen species (ROS). ROS, generated by a variety of enzymatic sources (such as mitochondria and NADPH oxidases, a.k.a. Nox), appear to play a pivotal role in vascular homeostasis, whereas elevated levels effect vascular disease. The pulmonary circulation is very sensitive to changes in the partial pressure of oxygen and differs from the systemic circulation in its response to this change. In fact, the pulmonary vessels contract in response to low oxygen tension, whereas systemic vessels dilate. Growing evidence suggests that ROS production and ROS-related pathways may be key factors that underlie this differential response to oxygen tension. A major emphasis of our laboratory is the role of Nox isozymes in cardiovascular disease. In this review, we will focus our attention on the role of Nox-derived ROS in the control of pulmonary vascular tone.

Disproportionate elevation of N-terminal pro-brain natriuretic peptide in scleroderma-related pulmonary hypertension.

N-terminal pro-brain natriuretic peptide (NT-proBNP) is a marker of neurohormonal activation that is useful in the diagnosis and prognosis of various forms of pulmonary arterial hypertension (PAH). We sought to characterise and compare NT-proBNP in a cohort of PAH related to systemic sclerosis (PAH-SSc) and idiopathic PAH (IPAH) patients. NT-proBNP levels, collected from PAH-SSc and IPAH patients followed prospectively, were compared and correlated with haemodynamic variables. Cox proportional hazard models were created to assess the predictive value of NT-proBNP. 98 patients (55 PAH-SSc, 43 IPAH) were included. Haemodynamics were similar, except for lower mean pulmonary arterial pressure in PAH-SSc. NT-proBNP levels were significantly higher in PAH-SSc (3,419+/-3,784 versus 1,393+/-1,633 pg x mL(-1); p<0.01) and were more closely related to haemodynamics in PAH-SSc than IPAH. 28 patients died. NT-proBNP predicted survival (hazard ratio (HR) 3.18; p<0.01) in the overall cohort; however, when stratified by group, predicted survival only in PAH-SSc (HR 3.07, p<0.01 versus 2.02, p = 0.29 in IPAH). This is the first description showing NT-proBNP levels are 1) significantly higher in PAH-SSc than IPAH despite less severe haemodynamic perturbations, and 2) stronger predictors of survival in PAH-SSc, suggesting that neurohormonal regulation may differ between PAH-SSc and IPAH. Future studies to define pertinent mechanisms are warranted.

Superoxide dismutase - a target for gene therapeutic approach to reduce oxidative stress in erectile dysfunction.

Erectile dysfunction (ED) is defined as the inability to attain and/or maintain penile erection sufficient for satisfactory sexual performance. Oxidative stress has been demonstrated to be involved in the pathophysiology of age- or diabetes-related ED. Superoxide dismutase (SOD), an antioxidant enzyme catalyzing the conversion of superoxide anion (O(2) (-)) to hydrogen peroxide (H(2)O(2)) and molecular oxygen (O(2)), is a promising therapeutic target for ED. In vivo gene therapy and adult stem cell-based ex vivo gene therapy are two attractive current gene therapies for the treatment of ED. In this chapter we describe the use of two potent gene transfer techniques to deliver the therapeutic gene extracellular superoxide dismutase (ecSOD) into the penis of aged or diabetic rats for therapy of ED: adenoviral-mediated intracavernosal ecSOD gene transfer for gene therapy of ED and ecSOD gene-modified marrow stromal cells, also known as mesenchymal stem cells, based stem cell and gene therapy.

Gene therapy techniques for the delivery of endothelial nitric oxide synthase to the lung for pulmonary hypertension.

Pulmonary hypertension (PH) is a serious, often fatal disease characterized by remodeling of the pulmonary vascular bed, increased pulmonary arterial pressure, and right heart failure. The increased vascular resistance in the pulmonary circulation is due to structural changes and increased vasoconstrictor tone. Although current therapies have prolonged survival, the long-term outcome is not favorable. Nitric oxide (NO) is synthesized by endothelial nitric oxide synthase (eNOS) and is important in regulating vascular resistance and in vascular remodeling in the lung. NO deficiency due to endothelial dysfunction plays an important role in the pathogenesis of PH. Therefore, local eNOS gene delivery to the lung is a promising approach for the treatment of PH. Adenoviral-mediated in vivo gene therapy and adult stem cell-based ex vivo gene therapy are two attractive current gene therapies for the treatment of cardiovascular and pulmonary diseases. In this chapter we describe the use of two gene transfer techniques, i.e., adenoviral gene transfer of eNOS and eNOS gene-modified rat marrow stromal cells, for eNOS gene delivery to the lung of laboratory animals for the treatment of PH.

Assessment of pulmonary vasculature and right heart by invasive haemodynamics and echocardiography.

Understanding the haemodynamical profile of the right ventricle and pulmonary circulation is critical to not only the initial evaluation of, but also the continued management of pulmonary hypertension. Despite advances in non-invasive imaging techniques, right heart catheterisation (RHC) remains the gold standard for diagnosis of pulmonary hypertension and its various causes. Even so, integration of invasive haemodynamical data with the echo-Doppler exam provides the most comprehensive assessment of the pathophysiology of pulmonary hypertension in the individual patient. Here, we review technical aspects of basic RHC as well as specialised procedures including exercise and fluid challenge in the evaluation of pulmonary hypertension. Interpretation of data in the context of pulmonary vascular disease is discussed. Echocardiographical assessment of the right ventricular structure and function in pulmonary vascular disease are discussed along with the integration of haemodynamical and echocardiographical data in the clinical context.

Right heart function and haemodynamics in pulmonary hypertension.

The primary challenge in the care of the patient with advanced pulmonary arterial hypertension (PAH) is right ventricular dysfunction with concomitant right heart failure. Right heart function is closely tied to survival in this disease, and there is a growing interest in the study of this unique structure. While echocardiography and cardiac magnetic resonance (CMR) have augmented our ability to image the right ventricle (RV), the primary means of assessing right heart function remains right heart catheterisation. Several of the currently available treatments for PAH have been shown to have effects on the RV, not just the pulmonary vasculature, and, in future, therapies aimed at optimizing right ventricular function may allow better outcomes in this challenging disease. New directions in right ventricular assessment including measurement of pulmonary vascular impedance and more widespread availability of CMR may allow greater knowledge about this little studied, yet highly important, right side of the heart.

Stem and endothelial progenitor cells in erection biology.

Erectile dysfunction (ED) is defined as the consistent inability to obtain or maintain an erection for satisfactory sexual relations. The past 20 years of basic science research on erection physiology has been devoted to investigating the pathogenesis of ED and has led to the conclusion that ED is predominately a disease of vascular origin with dramatic changes occurring in the endothelium. Research has also led to an understanding of the biochemical factors and intracellular mechanisms responsible for corporal smooth muscle contraction and relaxation and the influence of endothelium-derived relaxing factors. The development of methods to deliver both stem and endothelial cells to the penis has kindled a keen interest in treating ED with gene- and cell-based therapies. In this paper, erection physiology and stem cell biology is reviewed, and the potential application of novel cell-based therapies for the treatment of ED is discussed.

Altered high-energy phosphate metabolism predicts contractile dysfunction and subsequent ventricular remodeling in pressure-overload hypertrophy mice.

To study the role of early energetic abnormalities in the subsequent development of heart failure, we performed serial in vivo combined magnetic resonance imaging (MRI) and (31)P magnetic resonance spectroscopy (MRS) studies in mice that underwent pressure-overload following transverse aorta constriction (TAC). After 3 wk of TAC, a significant increase in left ventricular (LV) mass (74 +/- 4 vs. 140 +/- 26 mg, control vs. TAC, respectively; P < 0.000005), size [end-diastolic volume (EDV): 48 +/- 3 vs. 61 +/- 8 microl; P < 0.005], and contractile dysfunction [ejection fraction (EF): 62 +/- 4 vs. 38 +/- 10%; P < 0.000005] was observed, as well as depressed cardiac energetics (PCr/ATP: 2.0 +/- 0.1 vs. 1.3 +/- 0.4, P < 0.0005) measured by combined MRI/MRS. After an additional 3 wk, LV mass (140 +/- 26 vs. 167 +/- 36 mg; P < 0.01) and cavity size (EDV: 61 +/- 8 vs. 76 +/- 8 microl; P < 0.001) increased further, but there was no additional decline in PCr/ATP or EF. Cardiac PCr/ATP correlated inversely with end-systolic volume and directly with EF at 6 wk but not at 3 wk, suggesting a role of sustained energetic abnormalities in evolving chamber dysfunction and remodeling. Indeed, reduced cardiac PCr/ATP observed at 3 wk strongly correlated with changes in EDV that developed over the ensuing 3 wk. These data suggest that abnormal energetics due to pressure overload predict subsequent LV remodeling and dysfunction.

Addition of sildenafil to bosentan monotherapy in pulmonary arterial hypertension.

Combination therapy has been recommended for the treatment of pulmonary arterial hypertension (PAH). However, there is scant information on combination therapy after failure of monotherapy, particularly in patients with scleroderma-associated PAH (PAH-SSD). From a group of 82 consecutive patients with PAH who received initial bosentan monotherapy, a total of 13 idiopathic PAH (IPAH) and 12 PAH-SSD patients requiring additional therapy with sildenafil were studied. Sildenafil was added for clinical deterioration based upon symptoms, New York Heart Association (NYHA) classification or 6-min walk distance (6MWD). Clinical data and haemodynamics were collected at baseline. Assessments were made at 1-3-month intervals. At baseline, there were no differences in demographics, NYHA classification, haemodynamics or 6MWD between the two groups. After initiation of bosentan, both groups experienced clinical improvement but ultimately deteriorated (median time to monotherapy failure 792 versus 458 days for IPAH and PAH-SSD patients, respectively). After addition of sildenafil, more IPAH patients tended to improve in NYHA class (five out of 13 versus two out of 12) and walked further (mean difference in 6MWD 47+/-77 m versus -7+/-40 m) compared with PAH-SSD patients. In conclusion, addition of sildenafil after bosentan monotherapy failure improved New York Heart Association class and 6-min walk distance in idiopathic pulmonary arterial hypertension patients but failed to improve either parameter in scleroderma-associated pulmonary arterial hypertension patients. Additional studies are needed to assess the tolerability and efficacy of this combination in patients with scleroderma-associated pulmonary arterial hypertension.

Effect of combination endothelial nitric oxide synthase gene therapy and sildenafil on erectile function in diabetic rats.

Erectile dysfunction associated with diabetes mellitus is caused in part by disordered endothelial smooth muscle relaxation, neuropathy, and a decrease in cavernosal nitric oxide synthase (NOS) activity. The purpose of this study was to determine whether a combination of sildenafil and adenoviral gene transfer of endothelial NOS (eNOS) could enhance the erectile response in diabetic rats. Five groups of animals were utilized: (1) age-matched control rats, (2) streptozotocin (STZ)-induced diabetic rats (60 mg/kg i.p.), (3) STZ-rats + sildenafil (2 mg/kg i.v.), (4) STZ-rats transfected with AdCMVbetagal or AdCMVeNOS, and (5) STZ-rats transfected with AdCMVeNOS +sildenafil (2 mg/kg i.v.). At 2 months after i.p. injection of STZ, groups 4 and 5 were transfected with the adenoviruses and 1-2 days after transfection, all animals underwent cavernosal nerve stimulation (CNS) to assess erectile function. Cyclic 3',5'-guanosine monophosphate (cGMP) levels were assessed in the cavernosal tissue. STZ-diabetic rats had a significant decrease in erectile function as determined by the peak intracavernosal pressure (ICP) and total ICP (area under the erectile curve; AUC) after CNS when compared to control rats. STZ-diabetic rats+AdCMVeNOS had a peak ICP and AUC, which were similar to control animals. STZ-diabetic rats administered sildenafil demonstrated a significant increase in peak ICP at the 5 and 7.5 V settings, while the AUC was significantly increased at all voltage (V) settings. The increase in both ICP and AUC of STZ-diabetic rats transfected with AdCMVeNOS at all V settings was greater than STZ-diabetic rats transfected with AdCMVbetagal. STZ-diabetic rats transfected with AdCMVeNOS and administered sildenafil had a significant increase in total ICP that was greater than eNOS gene therapy alone. Cavernosal cGMP levels were significantly decreased in STZ-diabetic rats, but were increased after transfection with AdCMVeNOS to values greater than control animals. In conclusion, overexpression of eNOS and cGMP in combination with sildenafil significantly increased both the peak ICP and total ICP to CNS in the STZ-diabetic rat, which was similar to the response observed in control rats. Moreover, the total erectile response was greater in STZ-diabetic rats receiving eNOS gene therapy plus sildenafil than STZ-rats receiving sildenafil or eNOS gene therapy alone.

Peroxynitrite does not impair pulmonary and systemic vascular responses.

The effects of peroxynitrite (ONOO-) on vascular responses were investigated in the systemic and hindquarters vascular bed and in the isolated perfused rat lung. Intravenous injections of ONOO- decreased systemic arterial pressure, and injections of ONOO- into the hindquarters decreased perfusion pressure in a dose-related manner. Injections of ONOO- into the lung perfusion circuit increased pulmonary arterial perfusion pressure. Responses to ONOO- were rapid in onset, short in duration, and repeatable without exhibiting tachyphylaxis. Repeated injections of ONOO- did not alter systemic, hindquarters, or pulmonary responses to endothelium-dependent vasodilators or other vasoactive agonists and did not alter the hypoxic pulmonary vasoconstrictor response. Injections of sodium nitrate or nitrite or decomposed ONOO- had little effect on vascular pressures. Pulmonary and hindquarters responses to ONOO- were not altered by a cyclooxygenase inhibitor in a dose that attenuated responses to arachidonic acid. These results demonstrate that ONOO- has significant pulmonary vasoconstrictor, systemic vasodepressor, and vasodilator activity; that short-term repeated exposure does impair vascular responsiveness; and that responses to ONOO- are not dependent on cyclooxygenase product release.

Implications of nitric oxide synthase isoforms in the pathophysiology of Peyronie's disease.

Peyronie's disease is an idiopathic, localized connective tissue disorder of the penis which involves the tunica albuginea of the corpus cavernosum and the adjacent areolar space. Peyronie's disease is characterized by local changes in the collagen and elastic fiber composition of the tunica albuginea. The formation of fibrotic plaques alters penile anatomy and can cause different degrees of bending and narrowing, as well as penile pain and erectile dysfunction. Though long recognized as an important clinical entity of the male genitalia, the etiology of this disease has remained poorly understood. Until recently there have been no studies to examine the role nitric oxide (NO) and nitric oxide synthase (NOS) isoforms may play in the onset and progression of Peyronie's disease. NO is a potent biological mediator with diverse physiological and pathophysiological roles. The purpose of this review is to describe each of the NOS isoforms and their potential roles in the pathophysiology of Peyronie's disease, with particular emphasis on the regulation of endothelial and inducible NOS isoforms.

Differential responses to ATPgammaS in the mesenteric and hindlimb vascular bed of the cat.

The mechanism by which the purinergic agonist adenosine 5'-O-(3 thiotriphosphate) (ATPgammaS) decreases vascular resistance was investigated in the mesenteric and hindlimb vascular beds of the cat. Injections of ATPgammaS into the hindlimb perfusion circuit elicited dose-dependent decreases in perfusion pressure while injections into the mesenteric circuit produced a biphasic response with an initial vasopressor response followed by a vasodepressor response. In the mesenteric vascular bed the pressor response to ATPgammaS was blocked by a P2X1 receptor antagonist. Also an inhibitor of nitric oxide synthase enhanced the vasoconstrictive responses to ATPgammaS. However, the vasodepressor response in the mesenteric bed was not altered by the adminstration of an alpha adrenergic receptor antagonist, a cyclooxygenase inhibitor, a P2Y1 receptor antagonist, or a K+ATP channel blocking agent. These data suggest that the vasopressor response to ATPgammaS in the mesenteric vascular bed of the cat is mediated via P2X1 receptor activation. The differential responses to ATPgammaS in the hindlimb and mesentery suggest differences in purinergic receptor distribution in the vascular system of the cat. In addition, the results suggest that prostaglandin synthesis, P2Y1 receptor activation, alpha receptor inhibition, and K+ATP channels activation play little to no role in mediating the vascular response to ATPgammaS in the mesentery of the cat.

Vasodilator responses to ATP and UTP are cAMP dependent in the mesenteric vascular bed of the cat.

BACKGROUND: This study was designed to examine the responses to and the mechanism by which purinergic agonists decrease vascular resistance in the mesenteric vascular bed of the cat. METHODS AND RESULTS: Injections of ATP, UTP, and 2-MethylThioATP (2-MetSATP) into the mesenteric perfusion circuit elicited dose-dependent decreases in perfusion pressure while injections of beta,gamma-MethylATP (beta,gamma-MetATP) produced a biphasic response with an initial vasopressor response followed by a vasodilator response. The order of potency of the vasodilator response was 2-MetSATP > ATP > UTP > beta,gamma-MetATP. The vasodilator responses to ATP, UTP, 2-MetSATP, and beta,gamma-MetATP were increased in duration by the cAMP phosphodiesterase inhibitor, rolipram. However, vasodilator responses were not altered by the adminstration of a nitric oxide synthase inhibitor, a cGMP phosphodiesterase inhibitor, or a cyclooxygenase inhibitor. Treatment with PPADS, a P2X(1), P2Y(1), and P2Y(4) receptor antagonist, did not alter vasodilator responses to the purinergic agonists; however, the vasopressor component of the response to beta,gamma-MetATP was decreased. CONCLUSIONS: These data suggest that ATP, UTP, 2-MetSATP, and beta,gamma-MetATP dilate the mesentary vascular bed in the cat by a cAMP dependent mechanism, and that nitric oxide or prostaglandin release, cGMP accumulation, or activation of P2X(1), P2Y(1), or P2Y(4) receptors play little or no role in mediating vasodilator responses to the purinergic agonists in this regional vascular bed. In addition, these results suggest that the pressor component of the response to beta,gamma-MetATP is mediated by the activation of P2X(1) receptors.

Gene transfer of prepro-calcitonin gene-related peptide restores erectile function in the aged rat.

Erectile dysfunction in the aging male is caused, in part, by inadequate relaxation of the corpora cavernosal smooth musculature. Calcitonin gene-related peptide (CGRP), a peptide neurotrasmitter localized in the corpora cavernosa, is down-regulated in the aging rat penis. We examined the hypothesis that this reduction in CGRP may contribute to decreased cavernosal smooth muscle relaxation. Therefore, we sought to determine whether adenoviral-mediated gene transfer of prepro-CGRP (AdRSVCGRP) could enhance erectile responses in aged rats. We found a significant decrease in CGRP concentrations and in cAMP and cGMP levels in aged rat cavernosal tissue compared to younger rats. Aged rats also had significantly lower erectile function as determined by cavernosal nerve stimulation compared to younger rats. Five days after transfection with AdRSVCGRP, these aged rats had an approximately threefold increase in cavernosal CGRP levels compared to animals transfected with adenoviruses encoding nuclear-targeted beta-galactosidase (AdRSV beta gal). The AdRSVCGRP-transfected animals also demonstrated an increase in CGRP mRNA and immunohistochemical localization of CGRP in the smooth muscle of the corpora cavernosa. In addition, cAMP levels in the corpora cavernosa were significantly increased, whereas cGMP levels remained unchanged. Adenoviral transduction efficiency of beta-galactosidase reporter gene was measured by chemiluminescence and was observed in cavernosal tissue 5 days after transfection with AdRSV beta gal. More importantly, 5 days after administration of AdRSVCGRP, a significant increase was observed in the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of CGRP can physiologically improve erectile function in the aged rat.