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Background: B-type natriuretic peptide (BNP) immunoassays (BNP ia ) do not differentiate active and inactive forms. Inactive NT-proBNP is used to track heart failure (HF) during treatment with sacubitril/valsartan, which inhibits BNP degradation. Mass spectrometry (MS) may better assess effects of HF treatment on biologically active BNP1-32., Methods and Results: We developed a MS assay with immediate protease inhibition to quantify BNP1-32 over a linear range, using labeled recombinant BNP standard. In 4 healthy volunteers, BNP1-32 by MS (BNP MS ) increased from below the 5 pg/mL detection limit to 228 pg/mL after nesiritide. In patients with HF, BNP MS was measured in parallel with BNP and NT-proBNP immunoassays before and during sacubitril/valsartan treatment. BNP MS was 4.4-fold lower than BNP ia in patients with HF. Among patients not taking sacubitril/valsartan and without end-stage renal disease, BNP MS correlated with BNP ia (r s  = 0.77, P < .001) and NT-proBNP (r s  = 0.74, P < .001). After a median of 8 weeks on sacubitril/valsartan, active BNP MS levels decreased by 50% (interquartile range -98.3% to 41.7%, n = 22, P = .048) and correlated with NT-proBNP (r s  = 0.64, P < .001), but not with BNP ia (r s  = 0.46, P = .057)., Conclusions: Active BNP measured by MS accounts for only a small amount of BNP measured by immunoassays. Although decreased BNP production was anticipated to be masked by inhibition of degradation, levels of active BNP decreased during chronic sacubitril/valsartan treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: No prospectively derived or validated decision tools identify emergency department (ED) patients with acute heart failure (AHF) at low risk for 30-day adverse events who are thus potential candidates for safe ED discharge. This study sought to accomplish that goal., Background: The nearly 1 million annual ED visits for AHF are associated with high proportions of admissions and consume significant resources., Methods: We prospectively enrolled 1,033 patients diagnosed with AHF in the ED from 4 hospitals between July 20, 2007, and February 4, 2011. We used an ordinal outcome hierarchy, defined as the incidence of the most severe adverse event within 30 days of ED evaluation (acute coronary syndrome, coronary revascularization, emergent dialysis, intubation, mechanical cardiac support, cardiopulmonary resuscitation, and death)., Results: Of 1,033 patients enrolled, 126 (12%) experienced at least one 30-day adverse event. The decision tool had a C statistic of 0.68 (95% confidence interval: 0.63 to 0.74). Elevated troponin (p < 0.001) and renal function (p = 0.01) were significant predictors of adverse events in our multivariable model, whereas B-type natriuretic peptide (p = 0.09), tachypnea (p = 0.09), and patients undergoing dialysis (p = 0.07) trended toward significance. At risk thresholds of 1%, 3%, and 5%, we found 0%, 1.4%, and 13.0% patients were at low risk, with negative predictive values of 100%, 96%, and 93%, respectively., Conclusions: The STRATIFY decision tool identifies ED patients with AHF who are at low risk for 30-day adverse events and may be candidates for safe ED discharge. After external testing, and perhaps when used as part of a shared decision-making strategy, it may significantly affect disposition strategies. (Improving Heart Failure Risk Stratification in the ED [STRATIFY]; NCT00508638)., (Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Eosinophilic myocarditis is a rare condition in which inflammation of the heart results in an infiltrative cardiomyopathy that is often difficult to diagnose in the acute setting. It sometimes presents as left ventricular hypertrophy. The authors present a case of a 79-year-old woman with a history of Non-Hodgkin's lymphoma who presented with acute heart failure with marked left ventricular hypertrophy. Echocardiography demonstrated abnormalities consistent with an infiltrative cardiomyopathy, and endomyocardial biopsy showed findings consistent with eosinophilic myocarditis. The patient was managed with diuresis and glucocorticoid therapy, and within 4 weeks of her admission, her clinical status had improved and her echocardiogram normalized. The prompt diagnosis and treatment of this patient's myocarditis likely resulted in her favorable outcome. This illustrates the need for a broad consideration of all the potential causes of hypertrophy and the necessary diagnostic strategies and therapeutic options.

Background: Whole exome sequencing is a powerful technique for Mendelian disease gene discovery. However, variant prioritization remains a challenge. We applied whole exome sequencing to identify the causal variant in a large family with familial dilated cardiomyopathy of unknown pathogenesis., Methods and Results: A large family with autosomal dominant, familial dilated cardiomyopathy was identified. Exome capture and sequencing were performed in 3 remotely related, affected subjects predicted to share <0.1% of their genomes by descent. Shared variants were filtered for rarity, evolutionary conservation, and predicted functional significance, and remaining variants were filtered against 71 locally generated exomes. Variants were also prioritized using the Variant Annotation Analysis and Search Tool. Final candidates were validated by Sanger sequencing and tested for segregation. There were 664 shared heterozygous nonsense, missense, or splice site variants, of which 26 were rare (minor allele frequency ≤0.001 or not reported) in 2 public databases. Filtering against internal exomes reduced the number of candidates to 2, and of these, a single variant (c.1907 G>A) in RBM20, segregated with disease status and was absent in unaffected internal reference exomes. Bioinformatic prioritization with Variant Annotation Analysis and Search Tool supported this result., Conclusions: Whole exome sequencing of remotely related dilated cardiomyopathy subjects from a large, multiplex family, followed by systematic filtering, identified a causal RBM20 mutation without the need for linkage analysis.

Introduction: Significant advances have been made to understand the mechanisms involved in cardiac cell-based therapies. The early translational application of basic science knowledge has led to several animal and human clinical trials. The initial promising beneficial effect of stem cells on cardiac function restoration has been eclipsed by the inability of animal studies to translate into sustained clinical improvements in human clinical trials., Areas Covered: In this review, the authors cover an updated overview of various stem cell populations used in chronic heart failure. A critical review of clinical trials conducted in advanced heart failure patients is proposed, and finally promising avenues for developments in the field of cardiac cell-based therapies are presented., Expert Opinion: Several questions remain unanswered, and this limits our ability to understand basic mechanisms involved in stem cell therapeutics. Human studies have revealed critical unresolved issues. Further elucidation of the proper timing, mode delivery and prosurvival factors is imperative, if the field is to advance. The limited benefits seen to date are simply not enough if the potential for substantial recovery of nonfunctioning myocardium is to be realized.

Background: A critical challenge for physicians facing patients presenting with signs and symptoms of acute heart failure (AHF) is how and where to best manage them. Currently, most patients evaluated for AHF are admitted to the hospital, yet not all warrant inpatient care. Up to 50% of admissions could be potentially avoided and many admitted patients could be discharged after a short period of observation and treatment. Methods for identifying patients that can be sent home early are lacking. Improving the physician's ability to identify and safely manage low-risk patients is essential to avoiding unnecessary use of hospital beds., Methods: Two studies (STRATIFY and DECIDE) have been funded by the National Heart Lung and Blood Institute with the goal of developing prediction rules to facilitate early decision making in AHF. Using prospectively gathered evaluation and treatment data from the acute setting (STRATIFY) and early inpatient stay (DECIDE), rules will be generated to predict risk for death and serious complications. Subsequent studies will be designed to test the external validity, utility, generalizability and cost-effectiveness of these prediction rules in different acute care environments representing racially and socioeconomically diverse patient populations., Results: A major innovation is prediction of 5-day as well as 30-day outcomes, overcoming the limitation that 30-day outcomes are highly dependent on unpredictable, post-visit patient and provider behavior. A novel aspect of the proposed project is the use of a comprehensive cardiology review to correctly assign post-treatment outcomes to the acute presentation., Conclusions: Finally, a rigorous analysis plan has been developed to construct the prediction rules that will maximally extract both the statistical and clinical properties of every data element. Upon completion of this study we will subsequently externally test the prediction rules in a heterogeneous patient cohort., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Background: Galectin 3 (G3) is a mediator of fibrosis and remodeling in heart failure., Methods: Patients diagnosed with and treated for Acute Heart Failure Syndromes were prospectively enrolled in the Decision Making in Acute Decompensated Heart Failure multicenter trial., Results: Patients with a higher G3 had a history of renal disease, a lower heart rate and acute kidney injury. They also tended to have a history of HF and 30-day adverse events compared with B-type natriuretic peptide., Conclusion: In Acute Heart Failure Syndromes, G3 levels do not provide prognostic value, but when used complementary to B-type natriuretic peptide, G3 is associated with renal dysfunction and may predict 30-day events.

The authors sought to compare and contrast the clinical criterion standards currently used in a cohort of emergency department (ED) patients to diagnose acute heart failure syndromes (AHFS). In a prospective observational study of patients with signs and symptoms of AHFS, 3 criterion standards were examined: (1) the treating ED physician's diagnosis; (2) the hospital discharge diagnosis; and (3) a diagnosis based on medical record review by a panel of cardiologists. Using Cohen's kappa (κ) coefficient, the authors assessed agreement and then compared the different standards by repeatedly setting one as the criterion standard and the other two as index tests. A total of 483 patients were enrolled. Across all criterion standards, patients with AHFS were more likely to have a history of AHFS, congestion on physical examination and chest radiography, and elevated natriuretic peptide levels than those without AHFS. The standards agreed well (cardiology review vs hospital discharge diagnosis, κ=0.74; cardiology review vs ED diagnosis, κ=0.66; ED diagnosis vs hospital discharge diagnosis κ=0.59). Each method had similar sensitivity but differing specificities. Different criterion standards identify different patients from among those being evaluated for AHFS. Researchers should consider this when choosing between the various criterion standard approaches when evaluating new index tests., (© 2012 Wiley Periodicals, Inc.)

Heart failure requiring urgent therapy represents a burgeoning health care burden. Although acute heart failure syndromes are commonly defined as a change in chronic heart failure signs and symptoms requiring urgent therapy, the presentation, development, and response to treatment is highly dependent on individual patient characteristics. This heterogeneity has led to challenges in interpreting widely differing study methods, including eligibility requirements and outcome measures. To improve interpretation of results and translate such information to better patient care, it is essential to present an accurate description of the patient population and study design. Based on existing recommendations and expert consensus, the authors present standardized reporting criteria to improve interpretability of research in this challenging cohort., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: We evaluated the association of plasma uric acid alone and in combination with b-type natriuretic peptide (BNP) for emergency department (ED) diagnosis and 30-day prognosis in patients evaluated for acute heart failure (AHF)., Methods: We prospectively enrolled 322 adult ED patients with suspected AHF. Wilcoxon rank sum test, multivariable logistic regression and likelihood ratio (LR) tests were used for statistical analyses., Results: Uric acid's diagnostic utility was poor and failed to show significant associations with 30-day clinical outcomes. Uric acid also did not add significantly to BNP results., Conclusion: Among ED patients with suspected AHF, uric acid has poor diagnostic and prognostic utility.

Objective: Axillary intra-aortic balloon pump therapy has been described as a bridge to transplant. Advantages over femoral intra-aortic balloon pump therapy include reduced incidence of infection and enhanced patient mobility. We identified the patients who would benefit most from this therapy while awaiting heart transplantation., Methods: We conducted a single-center, retrospective observational study to evaluate outcomes from axillary intra-aortic balloon pump therapy. These included hemodynamic parameters, duration of support, and success in bridging to transplant. We selected patients on the basis of history of sternotomy, elevated panel-reactive antibody, and small body habitus. Patients were made to ambulate aggressively beginning on postoperative day 1., Results: Between September 2007 and September 2010, 18 patients underwent axillary intra-aortic balloon pump therapy. All patients had the devices placed through the left axillary artery with a Hemashield side graft (Boston Scientific, Natick, Mass). Before axillary placement, patients underwent femoral placement to demonstrate hemodynamic benefit. Duration of support ranged from 5 to 63 days (median = 19 days). There was marked improvement in ambulatory potential and hemodynamic parameters, with minimal blood transfusion requirements. There were no device-related infections. Some 72% of the patients (13/18) were successfully bridged to transplantation., Conclusions: Axillary intra-aortic balloon pump therapy provides excellent support for selected patients as a bridge to transplant. The majority of the patients were successfully bridged to transplant and discharged. Although this therapy has been described in previous studies, this is the largest series to incorporate a regimen of aggressive ambulation with daily measurements of distances walked., (Copyright © 2012. Published by Mosby, Inc.)

OBJECTIVES: We investigated the association of sST2 with diagnostic and prognostic outcomes and assessed whether it aids B-natriuretic peptide (BNP) in diagnosing and predicting outcomes in emergency department (ED) patients with suspected AHFS. METHODS: We recruited patients who presented to the ED of 3 tertiary hospitals with signs or symptoms of AHFS and met modified Framingham criteria for AHFS. Outcome measures were a final diagnosis of AHFS and 5-and 30-day adverse events. RESULTS: In the 295 subjects with sST2 available, the median sST2 was 0.20 ng/ml (IQR=0.10, 0.34). Although unadjusted analyses indicated sST2 was significantly associated with the diagnosis of AHFS (p=0.02), this was not so in the adjusted analysis (p=0.33). Moderately low diagnostic utility was noted with an AUC of 0.62 (95% CI=0.56, 0.69). Similar sST2 test characteristics were seen when BNP was restricted between 100 and 500 pg/ml. While sST2 was associated with AHFS readmission at 30-days (p=0.04), in the adjusted analyses it was not associated with adverse events. CONCLUSION: In patients with signs or symptoms of AHFS, unadjusted analyses indicated that sST2 was significantly associated with the diagnosis of AHFS and with 30-day AHFS recidivism. However, the associations did not carry over to adjusted analyses, and sST2 did not add significant information with regard to explaining the diagnostic and prognostic variability of BNP.

Hematopoietic stem cells (HSCs) are the most well-characterized and studied stem cells. They have been used to treat various benign and malignant hematologic disorders. Most stem cell transplant recipients survive more than 5 years without any evidence of their original clinical disease. Early animal trials have demonstrated the ability to improve cardiac function by transfer of HSCs into the myocardium, and early human studies have demonstrated the feasibility and safety of this approach. Trials in patients after myocardial infarction and with chronic heart failure have seen limited and mixed success, probably because of the various cell types and methods used., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Clinical trials involving novel therapies treating acute heart failure syndromes (AHFS) have shown limited success with regard to both efficacy and safety. As a direct result, outcomes have changed little over time and AHFS remains a disease process associated with largely no change in hospitalization rates (80%), hospital length of stay (median 4.5 days), and in-hospital (4-7%) and 60-day mortality (10%). Despite extensive emergency department (ED) involvement during the initial phase of AHFS management, clinical trials have enrolled patients after the ED phase of management, up to 48 hours after initial therapy, long after many patients have experienced significant beneficial effects of standard therapy. As standard therapy has provided symptomatic improvement in up to 70% of patients in these trials, it is not surprising that investigational agents started after 24 to 48 hours of standard therapy have shown limited clinical efficacy when compared with standard therapy., Methods and Results: The ability to screen, enroll, and randomize in the emergency setting is fundamental. The unique environment, the ethical complexities of enrollment in emergency-based research, and the need for rapid and standardized study-compliant care represent key challenges to active recruitment in AHFS studies. Specifically, the ability to identify and enroll a large cohort of AHFS patients early (<6 hours) in their presentation has been cited as the primary barrier to the appropriate design of clinical trials that includes this early window., Conclusions: In response, we have created a network of dedicated academic physicians with experience in clinical trials and acute management of heart failure who together can surmount this barrier and provide a framework for conducting early trials in AHFS.

The development of new antihypertensive agents is becoming even more important. We need better blood pressure control and also agents that treat hypertension as a disease of the vascular endothelium. Recently, it has been shown that blocking the renin-angiotensin system with angiotensin converting enzyme (ACE) inhibitors reduces blood pressure and decreases the incidence of vascular disease. Another peptide system, the natriuretic peptide system, has also been shown to be important in blood pressure control and volume homeostasis. Because ACE and neutral endopeptidase, the enzyme responsible for the degradation of the natriuretic peptides, are both zinc metalloproteases, new pharmaceuticals that inhibit both enzymes have been developed. The first of these, omapatrilat, has been shown to be an effective antihypertensive agent and to have great potential for treating congestive heart failure.

Guanosine 3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is required for this phosphorylation to occur. Vascular smooth muscle cells (VSMC) were used to determine if PDE5 is phosphorylated in intact cells when cGMP is increased. With the use of anti-PDE5 antibodies, a phosphorylated 93-kDa protein band was immunoprecipitated from early passaged primary cultures of VSMC that had been preincubated with 32(Pi) to label cellular ATP and then treated with atrial natriuretic factor (ANF). In the absence of ANF, there was no detectable incorporation of radiolabeled phosphate into this band. Phosphorylation of the 93-kDa protein was augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcGMP, which interacts poorly with the allosteric sites of PDE5, had no effect on PDE5 phosphorylation in the absence of ANF. Phosphorylation of PDE5 in response to treatment of cells with ANF was associated with a two- to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are deficient in PKG but retain PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing [gamma-32P]ATP resulted in 32(Pi) incorporation into PDE5 that was correlated with increased catalytic activity. These studies are the first to demonstrate phosphorylation of PDE5 in intact cells, thus suggesting a physiological role for this enzyme in smooth muscle regulation.

Previous work from this and other laboratories has demonstrated that the vasoconstrictor peptide angiotensin II results in hypertrophy of rat aortic smooth muscle cells that is associated with an increase in transcription of the early growth response gene c-fos. To explore the molecular mechanism responsible for c-fos induction in rat aortic smooth muscle cells, we used a series of reporter constructs linked to the chloramphenicol acetyl transferase gene in transient transfection experiments in rat aortic smooth muscle cells. Constructs containing both the serum response element and cAMP response element exhibited a 20-fold increase in chloramphenicol acetyl transferase activity in response to either serum or angiotensin II, whereas no increase was seen in vehicle-treated cells. Mutations in either the serum response element or cAMP response element alone, which have been demonstrated to inactivate these elements in other cell types, had no effect on chloramphenicol acetyl transferase inducibility. In contrast, if both elements were mutated, inducibility was almost abolished. Electrophoretic mobility shift assays with oligonucleotides corresponding to either serum response element or cAMP response element demonstrated that these oligonucleotides are capable of forming specific complexes with proteins from rat aortic smooth muscle cell nuclear extracts. One of the proteins binding to the serum response element is the previously described serum response factor, since it was supershifted by a monospecific antibody. These studies demonstrate that c-fox induction in smooth muscle occurs by a dual mechanism that can activate transcription via the serum response element or cAMP response element. These elements appear to act equally and independently, involving a distinct set of transacting factors.

Elevated levels of endogenous angiotensin can cause hypertensive nephrosclerosis as a result of the potent vasopressor action of the peptide. We have produced by gene targeting mice homozygous for a null mutation in the angiotensinogen gene (Atg-1-). Postnatally, Atg-1- animals show a modest delay in glomerular maturation. Although Atg-1- animals are hypotensive by 7 wk of age, they develop, by 3 wk of age, pronounced lesions in the renal cortex, similar to those of hypertensive nephrosclerosis. In addition, the papillae of homozygous mutant kidneys are reduced in size. These lesions are accompanied by local up-regulation of PDGF-B and TGF-beta1 mRNA in the cortex and down-regulation of PDGF-A mRNA in the papilla. The study demonstrates an important requirement for angiotensin in achieving and maintaining the normal morphology of the kidney. The mechanism through which angiotensin maintains the volume homeostasis in mammals includes promotion of the maturational growth of the papilla.

For both in vitro and in vivo experiments, especially in vascular cells, an efficient and easy method of gene transfer into this tissue would be extremely useful. Previous methods have either yielded low levels of expression or require complicated manipulation of viral vectors. The goal of this study was to develop an easy, efficient method to introduce unmodified plasmid DNA into vascular tissue. In this report it is demonstrated that complexing unmodified plasmid DNA with replication-deficient adenovirus (Ad5 dl312) via cationic lipids enhances gene transfer up to 1000-fold in cultured bovine aortic endothelial cells (BAECs). Further, utilizing a balloon-injured rabbit femoral artery model, intense nuclear staining in both the neointimal smooth muscle cell layer and the adventitia was seen following transfection with a plasmid containing the lacZ gene and the SV40 nuclear localization signal. Control arteries demonstrated no detectable staining. Our studies suggest that complexing plasmid DNA with adenovirus via lipids greatly enhances gene transfer both in vivo and in vitro. This method could have a wide range of applications for experiments in vascular tissue.

Background: Restenosis after catheter-based revascularization has been demonstrated to be primarily caused by medial and/or intimal smooth muscle cell (SMC) proliferation. The objective of this study was investigate the ability of local emission of beta-particles from a 32P-impregnated titanium "stent" wire source to inhibit vascular SMC and endothelial cell proliferation in cell culture and to determine the dose-response characteristics of this inhibition., Methods and Results: A series of experiments were performed using 0.20-mm-diameter titanium wires that were impregnated with varying low concentrations of 32P (activity range, 0.002 to 0.06 microCi/cm wire, n = 47) or 31P (nonradioactive control, n = 28) in cultures of rat and human aortic SMCs and in cultured bovine aortic endothelial cells. The zone of complete cell growth inhibition (in millimeters from stent wire) was measured using light microscopy in the cultures exposed to the radioactive (32P) or control (31P) wires at 6 and 12 days after plating. In both rat and human SMC cultures there was a distinct 5.5- to 10.6-mm zone of complete SMC inhibition at wire activity levels > or = 0.006 microCi/cm. In contrast, there was no zone of inhibition surrounding the control (31P impregnated) wires (P < .001 versus 32P wires at all wire activities > or = 0.006 microCi/cm for human and rat SMCs). Proliferating bovine endothelial cells were more radioresistant than SMCs, with no zone of inhibition observed at wire activity levels up to 0.019 microCi/cm (P < .001 versus SMCs at 0.006 microCi/cm and 0.019 microCi/cm)., Conclusions: We conclude that very low doses of beta-particle emission from a 32P-impregnated stent wire (activity levels as low as 0.006 microCi/cm of wire) completely inhibit the growth and migration of both rat and human SMCs within a range of 5.5 to 10.6 mm from the wire. Endothelial cells appear to be much more radioresistant than SMCs. These data suggest that an intra-arterial stent impregnated with a low concentration of 32P may have a salutary effect on the restenosis process. Whether this approach can be used successfully and safely to inhibit restenosis in vivo and in the clinical setting is under investigation.

In recent years numerous data have given evidence that the tissue renin-angiotensin system may play an equal or perhaps an even more important role than the circulating renin-angiotensin system in numerous physiologic processes. This was first suggested by the observation that the blood pressure lowering effect of angiotension-converting enzyme (ACE) inhibitors correlates better with tissue ACE activity than with plasma ACE activity. In response to hypertension and arterial injury, vascular smooth muscle cells undergo three responses: hypertrophy, hyperplasia, and remodeling. The end result is a decrease in lumen diameter and an increase in peripheral vascular resistance. Blockade of angiotensin II formation inhibits these smooth muscle responses in a number of animal models. This review discusses the evidence supporting the existence of local tissue renin-angiotensin system in the vasculature and its physiologic effects. Inhibition of the vascular renin-angiotensin system may have important implications in the treatment of patients with hypertension, atherosclerosis, and restenosis following balloon coronary angioplasty.

Our previous studies demonstrated that the sexually dimorphic pattern of hypertension in the spontaneously hypertensive rat is androgen dependent. Gonadectomy retards the development of hypertension in young males, but not in females, and administration of testosterone propionate to gonadectomized spontaneously hypertensive rats of both sexes confers a male pattern of blood pressure development. The current study tested the hypothesis that renal and hepatic renin and angiotensinogen gene expression are also androgen dependent in the spontaneously hypertensive rat. Male and female spontaneously hypertensive rats underwent gonadectomy or a sham operation at 4 weeks of age. Subgroups of gonadectomized rats of both sexes were implanted with a 15-mm or 30-mm Silastic capsule filled with testosterone at the same time the gonadectomy was performed; a third group received an empty Silastic capsule. Northern and slot blot analyses were used to characterize and quantitate renin and angiotensinogen messenger RNA (mRNA) in the kidney and liver 18 weeks after the gonadectomy. Blood pressure, plasma renin activity, and hepatic angiotensinogen mRNA levels were higher in intact males than in females. Orchidectomy retarded the development of hypertension and lowered plasma renin and renal and hepatic angiotensinogen mRNA levels, and testosterone replacement restored the male pattern of hypertension and plasma renin and increased renal and hepatic angiotensinogen mRNA. Ovariectomy did not alter blood pressure or plasma renin but did lower renal renin and renal and hepatic angiotensinogen mRNA; testosterone increased blood pressure, plasma renin, renal renin and angiotensinogen mRNA, and hepatic angiotensinogen mRNA levels in ovariectomized females.(ABSTRACT TRUNCATED AT 250 WORDS)

One of the major consequences of hypertension is an increase in the thickness of the arterial medial smooth muscle cell layer. This has been shown in both large and medium size resistance vessels caused by smooth muscle cell hypertrophy. Both in vivo and in vitro data suggest that the vasoconstrictor peptide angiotensin II (Ang II) may play an important role in the development of the smooth muscle hypertrophy. We have demonstrated that Ang II, when added to quiescence cultures of vascular smooth muscle cells, results in the rapid induction of the early growth response genes c-fos, c-myc, and c-jun. This is due to new transcription as demonstrated by nuclear runoff transcription assay, but is not dependent on new protein synthesis, as it is not blocked by the addition of cycloheximide. The effect is due, however, to an increase in intracellular calcium, suggesting that any vasoconstrictor which results in an increase in intracellular calcium may act in this manner. Following the induction of the early growth response genes there is delayed induction of the platelet derived growth factor A-chain gene. Data from our laboratory and from that of others has shown in preliminary studies that blockade of either the Ang II-induced increases in c-fos or in the platelet-derived growth factor A-chain increases smooth muscle cell protein synthesis. This suggests that Ang II and other vasoconstrictors may play an important role in vascular smooth muscle growth, in hypertension and also in atherosclerosis and following balloon injury of the arterial wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent data demonstrate the existence of a vascular renin angiotensin system. In this study we examine the localization of angiotensinogen mRNA in the blood vessel wall of two rat strains, the Wistar and Wistar Kyoto (WKY), as well as the regulation of vascular angiotensinogen mRNA expression by dietary sodium. Northern blot analysis and in situ hybridization histochemistry demonstrate that in both strains angiotensinogen mRNA is detected in the aortic medial smooth muscle layer as well as the periaortic fat. In WKY rats fed a 1.6% sodium diet, angiotensinogen mRNA concentration is 2.6-fold higher in the periaortic fat than in the smooth muscle, as analyzed by quantitative slot blot hybridization. Angiotensinogen mRNA expression in the medial smooth muscle layer is sodium regulated. After 5 d of a low (0.02%) sodium diet, smooth muscle angiotensinogen mRNA levels increase 3.2-fold (P less than 0.005) as compared with the 1.6% sodium diet. In contrast, angiotensinogen mRNA level in the periaortic fat is not influenced by sodium diet. In summary, our data demonstrate regional (smooth muscle vs. periaortic fat) differential regulation of angiotensinogen mRNA levels in the blood vessel wall by sodium. This regional differential regulation by sodium may have important physiological implications.

Angiotensin (Ang) II causes hypertrophy of rat aortic smooth muscle cells in culture and results in the rapid activation of c-fos. This study demonstrated that Ang II also activated c-jun and, in addition, could activate the AP-1 enhancer element. These data add support for a role of Ang II as an important mediator of vascular smooth muscle cell growth.

Because renin is an important enzyme in blood pressure regulation, we studied the possibility that an alteration in the structure of the human renin gene is genetically linked to human essential hypertension or associated with levels of plasma renin activity or blood pressure. By using specific DNA probes, we have identified four polymorphisms in the human renin gene with the restriction enzymes Taq I, HindIII, Bgl I, and Bgl II. The gene location of all of these polymorphisms except for the Bgl II polymorphism has been determined, and their frequencies were initially estimated in a population of 50 random subjects. To test the clinical significance of these polymorphisms, we studied 68 persons from a large Utah pedigree with a high incidence of hypertension. Among nine relatives with hypertension, genetic linkage without recombination was ruled out by observing several obligate recombinants. We also found no significant association of the restriction fragment length polymorphisms with quantitative measurements of sitting or standing, systolic or diastolic blood pressures, or plasma renin activity in 59 untreated members of this pedigree. Although we found no genetic linkage in this set of study subjects, the characterization of the restriction fragment length polymorphisms for the renin gene may be useful in future studies of other selected pedigrees for the presence of one or more of these to be a genetic marker in hypertension.

The effects of removing external calcium and inhibiting entry of calcium into the cell by treatment with D-600 on renin release from renal cortical slices of male Sprague-Dawley rats were examined. Baseline renin release, angiotensin II (AII)-induced inhibition, and isoproterenol-induced stimulation of renin release were studied. Removal of external calcium by chelation with 5 mM EGTA inhibited basal renin release while treatment with 1 mM EGTA stimulated basal renin release slightly. Incubation of slices with zero calcium medium containing 1 mM EGTA had no effect on isoproterenol-induced stimulation of renin release. In contrast, similar treatment reduced the inhibitory effect of AII from 58.7% of baseline to 85.3% (p less than 0.001). Similarly, blockage of calcium entry into cells with D-600 had no effect on isoproterenol-induced stimulation of renin release but abolished AII-induced inhibition. Replacement of sodium in the bathing medium with choline had no effect on baseline renin release or on AII-induced inhibition of renin release, ruling out the possibility that D-600 altered renin release via an effect on sodium influx. Taken together, the data strongly suggest that AII-induced inhibition of renin release is partially dependent on the presence of external calcium but that isoproterenol-induced stimulation of renin release is not.

In many experimental models, acute increases in sympathetic nervous system activity produce disproportionately greater vasoconstriction in the renal vascular bed than occurs in most other vascular beds. Since increased sympathetic nervous system activity has been implicated in the pathogenesis of DOCA-salt hypertension in the rat, we hypothesized that an attenuation of renal sympathetic tone would delay the development of this form of hypertension. Renal denervation was carried out in 5-week-old uninephrectomized male Sprague-Dawley rats 1 week before beginning DOCA-salt treatment. Systolic blood pressures using the tailcuff method in 32 sham-operated rats were significantly (p less than 0.05) elevated above control by Day five (115 +/- 3 vs 128 +/- 3 mm Hg) of DOCA-salt administration and continued to rise, reaching a plateau by Day 21 (192 +/- 5 mm Hg). In contrast, DOCA-salt administration in 32 renal denervated rats did not result in a significant elevation of blood pressure above control until Day 17 (121 +/- 3 vs 135 +/- 3 mm Hg, p less than 0.05). During the first 2 weeks of DOCA-salt treatment, fractional urinary sodium excretion was significantly greater (p less than 0.05) in renal denervated rats than in sham animals. During the third week of DOCA-salt administration, renal denervated rats had a rapid rise in blood pressure and a fall in fractional urinary sodium excretion to the level of the sham-operated animals. Coincident with the development of hypertension was a threefold increase in renal norepinephrine content (5.3 +/- 0.4 ng/g on Day 14 vs 17.7 +/- 3.0 ng/g on Day 24, p less than 0.01), suggesting reinnervation. These data suggest that increased renal sympathetic tone in the DOCA-salt rat facilitates sodium retention and is necessary for the development of the hypertension.

We previously observed that the renal nerves facilitate sodium retention and contribute to the development of DOCA-salt hypertension in the rat. To determine whether the renal nerves also participate in the maintenance of DOCA-salt hypertension, we studied the effects of renal denervation after 3 or 10 weeks of DOCA-salt treatment on systolic blood pressure, urinary sodium excretion, creatinine clearance, and precapillary arteriolar wall/lumen ratios of renal, hindlimb muscle, and cremaster muscle vascular beds. Systolic blood pressures of animals given DOCA-salt reached a plateau by 3 weeks of treatment at which time a sham operation or renal denervation was performed. Sham operation in hypertensive animals resulted in no change in systolic blood pressure and no change in percent sodium intake excreted. Wall/lumen ratio of the renal precapillary arteriole in sham-operated hypertensive animals was increased compared to similar sized vessels in hindlimb and cremaster muscle. In contrast, renal denervation resulted in a natriuresis and an attenuation of the hypertension (208 +/- 7 mmHg; p less than 0.01). Wall/lumen ratio of the renal capillary arterioles in renal denervated animals was no different than similar sized vessels in hindlimb and cremaster muscle and significantly less than that seen in sham-operated animals (0.85 +/- 0.05 vs 1.03 +/- 0.06; p less than 0.05). In another group of animals, sham operation or renal denervation was performed after 10 weeks of DOCA-salt treatment. At this time neither operation altered systolic blood pressure or sodium balance. In contrast to 3-week DOCA-salt-treated hypertensive sham-operated animals, renal precapillary arteriolar wall/lumen ratio of 10-week animals was no different than similar sized vessels in hindlimb and cremaster muscle. In addition, renal precapillary arteriolar wall/lumen ratio of 10-week DOCA-salt-treated renal-denervated animals was no different than that seen in 10-week DOCA-salt-treated sham-operated hypertensive animals. Creatinine clearance of the 10-week DOCA-salt-treated sham-operated or renal-denervated animals was significantly (p less than 0.01) lower than that of the 3-week DOCA-salt-treated groups (0.25 +/- 0.14 vs 1.03 +/- 0.10 ml/min). These data suggest that the renal nerves contribute to the early established phase of DOCA-salt hypertension by shifting the arterial pressure-renal sodium excretion curve to the right. With time, the renal nerves play a diminishing role in the maintenance of established DOCA-salt hypertension in the rat, while other renal factors, including decreased glomerular filtration rate and probable fixed renal vascular changes, play an increasingly important role.

The expression of the intrarenal renin angiotensin mRNAs in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were investigated in rats fed a low (0.02%)- or normal (1.6%)-sodium diet for 5 days. Total RNA was isolated from the kidneys and analyzed by both Northern and slot blots. The results indicated that the kidneys of all four groups of rats containing readibly detectable levels of renin and angiotensinogen mRNAs. The kidneys of the WKY contained higher levels of angiotensinogen mRNA under normal-salt diet compared with the SHR (P less than 0.01). Mild sodium depletion stimulated angiotensinogen mRNA in WKY kidneys by almost 50% compared with normal salt diet (P less than 0.01). In contrast, there was no significant difference in the renal angiotensinogen mRNA levels in the kidneys of SHR fed a low- or normal-sodium diet. Renin mRNA concentrations were comparable in both strains. Mild sodium depletion failed to yield any detectable changes in renin mRNA levels in either strain. Thus the SHR exhibits an alteration in the sodium regulation of intrarenal angiotensinogen mRNA expression. These results may have implications in the renal physiology of these animals.

To test the hypothesis that structural abnormalities exist in the cell membrane in persons with essential hypertension and that these abnormalities affect membrane-related cellular functions, we examined several membrane-dependent phenomena and membrane lipid composition in the blood cells of subjects with essential hypertension. We analyzed platelet aggregability, membrane fluidity, membrane fatty acid composition, and erythrocyte deformability in four normolipidemic subjects with untreated essential hypertension and in five age-matched normotensive controls. As compared with the controls, the subjects with essential hypertension had platelets that aggregated at lower concentrations of adenosine 5'-diphosphate, platelet membranes that were less fluid, and erythrocytes that were more deformable. Lipid analysis of the membranes of platelets from the two study groups showed that although the cholesterol content was identical, the membranes from the essential hypertension group contained significantly less linoleic acid (18:2) than did those from the normotensive controls. Given the known effects of cis-unsaturated fatty acyl composition on membrane fluidity and membrane-related cellular functions, these data suggest that one factor contributing to essential hypertension is an inherent structural membrane abnormality that alters the physical and functional properties of the cell membrane.

The mechanism and structural basis of the inhibition of renin release by angiotensin II (AII) were studied in rat kidney slices. Renin release was inhibited by AII and the (2-8), (3-8), (4-8), and (5-8) peptides of AII (5 X 10(-5) M). These constituent peptides of AII which share a common carboxyl terminus inhibited renin release with a sharp decrease in potency when the amino-terminal amino acid was removed. Saralasin attenuated the inhibition of renin release induced by equimolar concentrations of AII. Dose-response curves for AII and the (2-8) peptide [angiotensin III (AIII)] indicate that AII is a more potent inhibitor of renin release than is AIII. Depletion of renal norepinephrine by reserpine (10 mg/kg, i.p.) or pretreatment of slices with papaverine (1 X 10(-4) M) did not block the action of AII. The data give evidence for a direct action of AII on the juxtaglomerular cells independent of an interaction with either the sympathetic nervous system or the arteriolar baroreceptor and suggest that the intrarenal receptors that mediate AII-induced inhibition of renin release differ from AII receptors in the adrenal cortex.

The effects of altering sodium intake and inducing two-kidney, one-clip Goldblatt hypertension in the male Sprague-Dawley rat on renin release (RR) from renal cortical slices were examined. The low sodium diet significantly elevated plasma and kidney renin activity (KRA) and base-line renin release (BRR). KRA and BRR in the unclipped kidney of Goldblatt hypertensive rats were suppressed; KRA and BRR in the contralateral clipped kidney were comparable to values in normotensive animals fed a basal diet. Regression analysis of BRR vs. KRA for all kidneys studied yielded an r value of 0.728 for a linear model (y = a + bx) and 0.831 for a power model (y = axb), indicating that BRR is dependent on renal renin stores over a wide range of KRA, and suggesting that BRR may reach a maximal value. Percent inhibition of RR by angiotensin II (AII) and percent stimulation of RR by isoproterenol were the same in high, low, and basal salt states, while absolute changes in RR and changes in RR expressed as percent of renin stores released per hour were less in the high salt state, suggesting that isoproterenol-induced stimulation and AII-induced inhibition of RR are dependent on renal renin stores.

Angiotensin II (Ang II) has been shown to cause hypertrophy of cultured quiescent rat aortic smooth muscle cells. This observation along with the recent demonstration of angiotensinogen messenger RNA (mRNA) in the vessel wall has led us to postulate a role for Ang II in hypertensive smooth muscle hypertrophy. One of the earliest responses in a wide variety of cells in response to a growth-promoting agent is the induction of the proto-oncogene c-fos. To investigate the mechanism of the action of Ang II, we investigated the effect of Ang II on the expression of the c-fos gene in rat aortic smooth muscle cells that were made quiescent by being grown in a defined serum-free media for 48 hours. Ang II (10(-6)-10(-10) M) resulted in a dose-dependent increase in c-fos mRNA expression. This induction was angiotensin-receptor specific since it was completely abolished by the competitive inhibitor saralasin. Inhibition of protein synthesis did not block the rise in c-fos mRNA expression; it resulted in a superinduction and stabilization of the c-fos mRNA. Using a nuclear runoff transcription assay, we demonstrated that Ang II stimulated the transcription rate of the c-fos gene. This activation of c-fos gene expression may be an important mechanism in the angiotensin-induced smooth muscle hypertrophy.

Recently, angiotensin II (Ang II) has been shown to cause hypertrophy of cultured quiescent rat aortic smooth muscle (RASM) cells. This observation along with the demonstration of angiotensinogen mRNA in the vessel wall has led us to postulate a role for vascular angiotensin in hypertensive blood vessel hypertrophy. To investigate further the possible molecular mechanisms, we examined the effect of Ang II on the expression of two genes known to be involved with cellular growth response. Near-confluent RASM cells were made quiescent by 48-h exposure to a defined serum-free medium. Ang II (10(-6) to 10(-11) M) resulted in an induction of the protooncogene c-myc mRNA within 30 min which persisted for 6 h. Interestingly, 6 h after the addition of Ang II, platelet-derived growth factor (PDGF) A-chain mRNA expression was elevated, peaked in 9 h, and persisted for 11 h. This was accompanied with a 15-20-fold increase in PDGF concentration in the culture medium. These effects were dose-dependent and were blocked by saralasin. Whereas the inhibition of protein synthesis by cycloheximide resulted in a stabilization of c-myc mRNA, cycloheximide abolished the elevation of the PDGF A-chain mRNA. Taken together, our data show that exposure of RASM cells to Ang II results in the sequential activation of c-myc and PDGF A-chain mRNA expressions. This sequential activation of protooncogene and growth factor gene may be an important mechanism in angiotensin-induced smooth muscle growth and hypertrophy.

Probes to hypervariable minisatellite regions of DNA identify multiple loci scattered over the autosomal chromosomes and produce a complex Southern blot pattern of fragments termed a DNA 'fingerprint'. As concern has been raised that different stocks of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) may not be biologically identical, we have compared the DNA of SHR and WKY from several sources using two such probes which identify different sets of minisatellite sequences. While the DNA fingerprints of SHR from the various sources were identical, variability was observed in those of WKY, indicating genetic heterogeneity between different WKY stocks. In animals from one of the commercial suppliers even inter-rat variability in DNA fingerprints was seen, suggesting genetic heterogeneity within that single colony. These observations indicate that experimental results obtained using WKY from different sources may not be directly comparable and could provide an explanation for some of the conflicting data that exist on the comparative characteristics of SHR and WKY. In separate studies, direct comparisons both of the DNA fingerprints of SHR and WKY and of SHR and stroke-prone spontaneously hypertensive rats (SHRSP) showed multiple differences between the strains. The polymorphisms seen could provide useful linkage markers in locating the chromosomal sites of the genetic loci responsible for raised blood pressure in the SHR and the propensity to strokes in the SHRSP.