Paul W. Wennberg

Presence of Dendroaspis natriuretic peptide-like immunoreactivity in human plasma and its increase during human heart failure

OBJECTIVE To determine whether Dendroaspis natriuretic peptide (DNP), a novel peptide isolated from the venom of the Dendroaspis angusticeps snake that contains a 17-amino acid disulfide ring structure similar to that in atrial, brain, and C-type natriuretic peptides, is present in normal human plasma and myocardium and whether, like the other natriuretic peptides, DNP-like immunoreactivity (DNP-LI) is activated in human congestive heart failure (CHF). MATERIAL AND METHODS Circulating DNP-LI was assessed in 19 normal human subjects and 19 patients with CHF (New York Heart Association class III or IV) with a specific and sensitive radioimmunoassay for DNP with no cross-reactivity with the other natriuretic peptides. Immunohistochemical studies that used polyclonal rabbit anti-DNP antiserum were performed on human atrial myocardial tissue obtained from four patients with end-stage CHF who were undergoing cardiac transplantation and from three donor hearts at the time of transplantation. RESULTS We report that DNP-LI circulates in normal human plasma and is present in the normal atrial myocardium. In addition, DNP-LI is increased in the plasma of patients with CHF. CONCLUSION This study demonstrates, for the first time, the presence of a DNP-like peptide in normal human plasma and in the atrial myocardium. Additionally, these studies demonstrate increased plasma DNP-LI in human CHF. These results support the possible existence of an additional new natriuretic peptide in humans, which may have a role in the neurohumoral activation that characterizes human CHF.

Successful iliac vein and inferior vena cava stenting ameliorates venous claudication and improves venous outflow, calf muscle pump function, and clinical status in post-thrombotic syndrome.

Objectives:Stent therapy has been proposed as an effective treatment of chronic iliofemoral (I-F) and inferior vena cava (IVC) thrombosis. The purpose of this study was to determine the effects of technically successful stenting in consecutive patients with advanced CVD (CEAP3–6 ± venous claudication) for chronic obliteration of the I-F (±IVC) trunks, on the venous hemodynamics of the limb, the walking capacity, and the clinical status of CVD. These patients had previously failed to improve with conservative treatment entailing compression and/or wound care for at least 12 months. Methods:The presence of venous claudication was assessed by ≥3 independent examiners. The CEAP clinical classification was used to determine the severity of CVD. Outflow obstruction [Outflow Fraction at 1- and 4-second (OF1 and OF4) in %], venous reflux [Venous Filling Index (VFI) in mL/100 mL/s], calf muscle pump function [Ejection Fraction (EF) in %] and hypertension [Residual Venous Fraction (RVF) in %], were examined before and after successful venous stenting in 16 patients (23 limbs), 6 females, 10 males, median age 42 years; range, 31–77 yearas, left/right limbs 14/9, using strain gauge plethysmography; 7/16 of these had thrombosis extending to the IVC. Contralateral limbs to those stented without prior I-F ± IVC thrombosis, nor infrainguinal clots on duplex, were used as control limbs (n = 9). Excluded were patients with stent occlusion or stenoses, peripheral arterial disease (ABI <1.0), symptomatic cardiac disease, unrelated causes of walking impairment, and malignancy. Preinterventional data (≤30 days) were compared with those after endovascular therapy (8.4 months; interquartile range [IQR], 3–11.8 months). Nonparametric analysis was applied. Results:Compared with the control group, limbs with I-F ± IVC thrombosis before stenting had reduced venous outflow (OF4) and calf muscle pump function (EF), worse CEAP clinical class, and increased RVF (all, P < 0.05). At 8.4 months (IQR, 3–11.8 months) after successful I-F (±IVC) stenting, venous outflow (OF1, OF4) and calf muscle pump function (EF) had both improved (P < 0.001) and the RVF had decreased (P < 0.001), at the expense of venous reflux, which had increased further (increase of median VFI by 24%; P = 0.002); the CEAP status had also improved (P < 0.05) from a median class C3 (range, C3–C6; IQR, C3–C5) [distribution, C6: 6; C4: 4; C3: 13] before intervention to C2 (range, C2–C6; IQR, C2–C4.5) [distribution, C6: 1; C5: 5; C4: 4; C2: 13] after intervention. At this follow up (8.4 months median), venous outflow (OF1, OF4), calf muscle pump function (EF), and RVF of the stented limbs did not differ significantly from those of the control; significantly worse (P < 0.025) were the amount of venous reflux (VFI), and the CEAP clinical class, despite the improvement with stenting. Incapacitating venous claudication noted in 62.5% (10 of 16, 95% CI, 35.8%–89.1%) of patients (15 of 23 limbs; 65.2%, 95% CI, 44.2%–86.3%) before stenting was eliminated in all after stenting (P < 0.001). Conclusions:Successful I-F (±IVC) stenting in limbs with venous outflow obstruction and complicated CVD (C3–C6) ameliorates venous claudication, normalizes outflow, and enhances calf muscle pump function, compounded by a significant clinical improvement of CVD. The significant increase in the amount of venous reflux of the stented limbs indicates that elastic or inelastic compression support of the successfully stented limbs would be pivotal in preventing disease progression.

Vascular Actions of Brain Natriuretic Peptide: Modulation by Atherosclerosis and Neutral Endopeptidase Inhibition

OBJECTIVES We sought to define the vascular actions of the cardiac hormone brain natriuretic peptide (BNP) on cellular proliferation and cyclic guanosine monophosphate (cGMP) in human aortic vascular smooth muscle cells (HAVSMCs). Secondly, we investigated BNP and acetylcholine (ACh) vasorelaxations in aortic rings from normal and atherosclerotic rabbits in the presence and absence of long-term oral inhibition of neutral endopeptidase (NEP). BACKGROUND The vascular actions of BNP are not well defined, despite the presence of its receptor in vascular smooth muscle and the upregulation of NEP, the ectoenzyme that degrades BNP, in the vascular wall in atherosclerosis. METHODS HAVSMCs stimulated with fetal calf serum (FCS) were pulsed with bromodeoxyuridine (BrdU) with and without BNP. The HAVSMCs were incubated in the presence and absence of BNP to assess cGMP. Vasorelaxations to BNP and ACh were assessed in rings in normal and atherosclerotic rabbits in the presence and absence of long-term oral inhibition of NEP, together with assessment of atheroma formation. RESULTS FCS-stimulated BrdU uptake in HAVSMCs was suppressed with BNP. BNP potentiated cGMP in HAVSMCs. BNP resulted in potent vasorelaxation in normal isolated aortic rings, which were impaired in atherosclerotic versus normal rabbits and preserved with NEP inhibition, which also decreased atheroma formation. Relaxations to ACh, which were also impaired in atherosclerosis, were preserved with inhibition of NEP. CONCLUSIONS We conclude that BNP potently inhibits vascular smooth muscle cell proliferation and potentiates the generation of cGMP. BNP potently relaxes the normal rabbit aorta, and this response is impaired in atherosclerosis but preserved with inhibition of NEP, together with a reduction in atheroma formation and preservation of relaxations to ACh.

Modulation of Functionally Active Endothelin-Converting Enzyme by Chronic Neutral Endopeptidase Inhibition in Experimental Atherosclerosis

BACKGROUND Endothelin-converting enzyme-1 (ECE-1) processes big endothelin-1 (ET-1) to ET-1, a peptide implicated in atheroma formation. ECE-1 exists in 2 isoforms (ECE-1alpha and ECE-1beta), the result of alternative splicing of a common gene. Neutral endopeptidase (NEP) is a genetically distinct metallopeptidase that degrades the natriuretic peptides. These peptides mediate antiproliferative and vasodilating actions. We sought to demonstrate the distribution of the 2 ECE-1 isoforms in experimental atherosclerosis, to determine the effects of chronic NEP-I on plasma cGMP concentrations, vascular wall ECE-1 activity, and ET-1 concentration, and to correlate these actions with atheroma formation. Our hypothesis was that chronic NEP-I, in association with augmented cGMP, would inhibit ECE-1 conversion of big ET-1 to active ET-1, thus reducing tissue ET-1 concentrations and associated atheroma formation. METHODS AND RESULTS Cholesterol-fed New Zealand White rabbits (n=8, 1% cholesterol diet) and NEP-I-treated cholesterol-fed New Zealand White rabbits (n=8; candoxatril, 30 mg/kg per day, Pfizer) were euthanized after 8 weeks of feeding. ECE-1alpha and ECE-1beta immunoreactivity was present in the aortas of both groups. Compared with control values, plasma cGMP concentrations were increased (2.8+/-0.6 versus 8.4+/-1.2 pmol/mL, P<0.05), ECE-1 activity was attenuated (68+/-3% versus 32+/-8%, P<0. 05), aortic tissue ET-1 concentrations were reduced (4.6+/-0.5 versus 3.2+/-0.3 pg/mg protein, P<0.05), and atheroma formation was attenuated (62+/-6% versus 34+/-5%, P<0.01) by NEP-I. CONCLUSIONS These studies suggest that ECE-1 is present and functionally active in the vascular wall in atherosclerosis. Inhibition of ECE-1 by NEP-I represents a novel approach to interruption of the endothelin system in this cardiovascular disease state.

Disease location is associated with survival in patients with peripheral arterial disease.

Background We investigated whether disease location influences survival in patients with peripheral arterial disease. Methods and Results Patients (n=12 731; mean age, 67.5±12.7 years; 57.4% male) who underwent outpatient noninvasive lower extremity arterial evaluation were followed up for 5.9±3.1 years for all‐cause mortality. Peripheral arterial disease (n=8930) was defined as a resting or postexercise ankle‐brachial index (ABI) ≤0.90, and normal ABI (n=3 801) was defined as a resting and postexercise ABI of 1.00 to 1.30. Presence or absence of disease at the proximal location or distal location was determined on the basis of Doppler signals in leg arteries; 42% had no PD or DD, 45% had proximal (14% postexercise PD only), 30% had distal disease, 17% had both proximal and distal disease, 28% had proximal only and 14% had distal only. We performed multivariable logistic regression to identify factors associated with disease location, and Cox proportional hazard regression to assess the respective effects of proximal or distal disease on survival. Older age, male sex, diabetes, heart failure, and critical limb ischemia were associated with distal disease, whereas female sex, smoking, hypertension, dyslipidemia, coronary heart disease, cerebrovascular disease, chronic obstructive pulmonary disease, and critical limb ischemia were associated with proximal disease. Over a mean follow‐up of 5.9±3.1 years, 3039 patients (23.9%) died. After adjustment for potential confounders, the hazard ratios (HRs) of death associated with PD only and DD only were 1.3 (1.3 to 1.4) and 1.5 (1.4 to 1.6), respectively. After additional adjustment for resting ABI, there was no significant association between proximal disease and death, whereas the association of distal disease with death remained significant (HR, 1.2; 95% CI, 1.1 to 1.3). Conclusions In patients with peripheral arterial disease, proximal and distal disease locations were associated with distinctive risk factor and comorbidity profiles. Distal disease was associated with worse survival even after adjustment for risk factors, comorbidities, and resting ABI.

Further attenuation of endothelium-dependent relaxation imparted by natriuretic peptide receptor antagonism.

Nitric oxide (NO) is an important endothelium-derived relaxing factor that functions via activation of soluble guanylyl cyclase and cGMP generation in vascular smooth muscle. Recently, studies have described the synthesis and secretion of C-type natriuretic peptide (CNP) from endothelial cells. This peptide also mediates relaxation via cGMP but through activation of particulate guanylyl cyclase. We tested the hypothesis that endothelium-dependent relaxations to acetylcholine or bradykinin in isolated canine coronary arteries involve both releases of NO and CNP. Rings of canine coronary arteries were incubated with either inhibitors of NO production ( N G-monomethyl-l-arginine,l-NMMA) or the natriuretic peptide receptor antagonist HS-142-1. CNP caused concentration-dependent relaxations of rings with and without endothelium. These relaxations were attenuated by HS-142-1. Relaxations to acetylcholine and bradykinin were attenuated byl-NMMA alone but not attenuated by HS-142-1 alone. Coinhibition withl-NMMA and HS-142-1 significantly inhibited acetylcholine- and bradykinin-induced relaxation to a magnitude greater than either inhibitor alone. In summary, a novel interaction between the NO and the natriuretic peptide system is demonstrated by increased attenuation of endothelium-dependent relaxations to acetylcholine and bradykinin when both NO synthase and natriuretic peptide receptors are inhibited. These investigations support the concept of release of multiple endothelium-derived factors in response to acetylcholine- and bradykinin-receptor stimulation in endothelial cells, which may include CNP, as well as NO.

Enhanced endothelin-converting enzyme immunoreactivity in early atherosclerosis.

Endothelin-1 (ET-1) is a 21-amino-acid local and circulating factor whose plasma concentrations are increased in advanced atherosclerosis. ET-1 is cleaved from a prohormone (big ET-1) by endothelin-converting enzymes (ECEs) into the biologically active mature form which mediates vasoconstriction and cell proliferation. This study was designed to test by immunohistochemistry the hypothesis that ECE is present locally in the neointima of atherosclerotic vessels. Two groups of rabbits, control (n = 6) and cholesterol-fed (1% cholesterol diet for 8 weeks; n = 6) were sacrificed. Aortas were excised and divided for determination of tissue ET-1 concentration by RIA and immunohistochemical analysis of ECE. Vascular wall ET-1 was increased in the atherosclerotic aorta (6.1 +/- 0.8 vs. 9.8 +/- 0.9 pg/mg protein; p < 0.05), whereas circulating ET-1 concentrations were similar in the two groups (3.8 +/- 0.4 vs. 2.4 +/- 1.4 pg/ml). Immunostaining revealed the presence of ECE in endothelial and vascular smooth-muscle cells of the control group. Enhanced ECE immunoreactivity was present in atherosclerotic aortas, particularly in the neointimal macrophages and smooth-muscle cells. We conclude that local vascular wall, but not circulating ET-1, is increased in early atherosclerosis. In addition, ECE immunoreactivity is increased in early atherosclerosis and may therefore contribute to the generation of local ET-1 in early experimental atherosclerosis. These studies provide important insights into the regulation of ET-1 in early atherosclerosis, which may contribute to the elucidation of factors involved in the progression of atherosclerosis.

Approach to the Patient With Peripheral Arterial Disease

How best to approach a patient with peripheral arterial disease (PAD) can be an intimidating and confusing task. There are few disease processes as variable in location, presentation, and severity as those seen in the vasculopath. There are many etiologies to be considered simultaneously. Ironically, it is the variability of PAD that provides an opportunity to extract a comprehensive and tightly integrated history and physical examination that is nearly unparalleled in medicine. Similarly, there are few disease processes that offer the opportunity to impact so many aspects of the patient, literally from head to toe. This article, part of a series on PAD, focuses on the initial assessment and evaluation of the PAD, with pathophysiology, genetics, and treatment addressed in accompanying articles. Each patient with PAD is unique (Table 1). Even though the pathophysiology, risk factors, location, and eventual treatment options for a patient often prove routine, the manner in which the patient presents is anything but predictable.1 We have all had the experience of taking a well-structured history (Table 2) from a textbook patient, presented to an attending physician or a colleague, and then watched and listened in disbelief as the confirmatory history is nothing at all similar to what was heard 15 minutes earlier. Unfortunately, patients do not often read the textbook. As demonstrated by McDermott and colleagues,2 the discomfort caused by PAD is more often atypical than typical. Descriptions such as “tired,” “giving way,” “sore,” and “hurts” are offered more often than “cramp.” This forces the healthcare provider to meticulously clarify the location, quality, and circumstances of the discomfort. It is not unusual that a patient reports ≥2 types and locations of discomfort. Careful consideration for pain from both orthopedic and neurogenic sources must be taken. Each symptom should be individually addressed and clarified. These …

Discordant Diagnosis of Lower Extremity Peripheral Artery Disease Using American Heart Association Postexercise Guidelines

Abstract To determine whether postexercise criteria for peripheral artery disease (PAD) diagnosis recommended by the American Heart Association (AHA) identifies the same group of PAD patients. Diagnosis of PAD is performed using ankle-brachial index at rest (resting-ABI). When resting-ABI is not contributive, an AHA scientific statement recommend to use 1 of 2 following criteria: a postexercise ABI decrease of greater than 20% or a postexercise ankle pressure decrease of greater than 30 mm Hg. Between 1996 and 2012, 31,663 consecutive patients underwent lower-extremity arterial study at Mayo Clinic. Among them, only unique patients who had exercise treadmill testing were analyzed. In this retrospective analysis, resting-ABI, postexercise ABI, and postexercise decrease of ankle pressure measured at 1-minute were measured in each patient. We conducted an analysis of agreement between postexercise criteria expressing the agreement separately for the positive and the negative ratings. Twelve thousand three hundred twelve consecutive patients were studied with a mean age of 67 ± 12 years, 61% male. According to resting-ABI, 4317 (35%) patients had PAD. In the whole population, if a clinician diagnoses “PAD” with 1 postexercise criterion, the probability that other clinicians would also diagnose “PAD” is 74.3%. If a clinician diagnoses “no PAD”, the probability that other clinicians would also diagnose “no PAD” is 82.4%. In the patients to be of potential benefit from treadmill test when the resting-ABI > 0.90, if a clinician diagnoses “PAD” with 1 postexercise criterion, the probability that other clinicians would also diagnose “PAD” is 58.4% whereas if a clinician diagnoses “no PAD,” the probability that other clinicians would also diagnose “no PAD” is 87.5%. Postexercise criteria do not identify the same group of PAD patients. In our opinion, postexercise criteria to define PAD deserve additional study.

Application of exercise transcutaneous oxygen pressure measurements for detection of proximal lower extremity arterial disease: A case report.

Proximal claudication is secondary to ischemia caused by peripheral artery disease (PAD), whereas proximal pseudo-claudication is secondary to other disease processes such as hip arthritis, spinal stenosis, neuropathy, and so forth. The differentiation between the two can be challenging. Exercise transcutaneous oxygen pressure measurement (exercise-TcPO2) allows noninvasive detection of flow-reducing lesions in the proximal arteries and tributaries of the lower extremity arterial tree. We present the first case report in the United States using an exercise-TcPO2 algorithm. A 71-year-old diabetic patient with proximal left-sided and right-calf claudication with indeterminate ankle-brachial indices underwent an exercise-TcPO2 study before and after endovascular intervention. Four TcPO2 probes were placed: one at chest level (reference probe), one on each buttock, and one on the symptomatic calf. The Delta from Resting Oxygen Pressure (DROP) index was calculated at each probe site using a previously validated protocol. Proximal left- and right-calf ischemia were confirmed by the initial exercise-TcPO2, and, after endovascular treatment of the left iliac artery lesion, improvements in proximal exercise-TcPO2 values were found. These data suggest that exercise-TcPO2 can be useful in PAD evaluation in patients with non-compressible arteries and/or proximal claudication.