Placental senescence pathophysiology is shared between peripartum cardiomyopathy and preeclampsia in mouse and human.

Peripartum cardiomyopathy (PPCM) is an idiopathic form of pregnancy-induced heart failure associated with preeclampsia. Circulating factors in late pregnancy are thought to contribute to both diseases, suggesting a common underlying pathophysiological process. However, what drives this process remains unclear. Using serum proteomics, we identified the senescence-associated secretory phenotype (SASP), a marker of cellular senescence associated with biological aging, as the most highly up-regulated pathway in young women with PPCM or preeclampsia. Placentas from women with preeclampsia displayed multiple markers of amplified senescence and tissue aging, as well as overall increased gene expression of 28 circulating proteins that contributed to SASP pathway enrichment in serum samples from patients with preeclampsia or PPCM. The most highly expressed placental SASP factor, activin A, was associated with cardiac dysfunction or heart failure severity in women with preeclampsia or PPCM. In a murine model of PPCM induced by cardiomyocyte-specific deletion of the gene encoding peroxisome proliferator-activated receptor γ coactivator-1α, inhibiting activin A signaling in the early postpartum period with a monoclonal antibody to the activin type II receptor improved heart function. In addition, attenuating placental senescence with the senolytic compound fisetin in late pregnancy improved cardiac function in these animals. These findings link senescence biology to cardiac dysfunction in pregnancy and help to elucidate the pathogenesis underlying cardiovascular diseases of pregnancy.

Association of activin A and postpartum blood pressure in peripartum cardiomyopathy.

BACKGROUND: Activin A has been implicated in the pathogenesis of patients with chronic hypertension and heart failure as well as patients with hypertensive disorders of pregnancy (HDP). Whether activin A correlates with blood pressure in patients with peripartum cardiomyopathy (PPCM) and HDP history has not previously been explored. METHODS AND RESULTS: 82 women with PPCM w/ and w/out HDP or hypertension history were selected for analysis from the Investigations in Pregnancy Associated Cardiomyopathy (IPAC) study. Serum biomarkers and blood pressure were assessed at the time of enrollment (median postpartum day 24). Levels of both sFlt-1 (SBP: r 0.47, p = 0.008; DBP: r 0.57, p < 0.001) and activin A (SBP: r 0.59, p < 0.001;DBP: r 0.68, p < 0.001) were noted to significantly correlate with blood pressure in patients with a history of HDP who went on to develop PPCM, but not in patients with chronic hypertension or no hypertensive history. The strongest correlation was between activin A levels and postpartum diastolic blood pressure for the subset with preeclampsia (DBP: r0.82, p < 0.001). This remained significant in multivariable linear regression analysis (DBP: ß = 0.011, p = 0.015). CONCLUSION: In patients with PPCM, activin A and sFlt-1 levels had direct correlations with both systolic (SBP) and diastolic blood pressures (DBP), but only in participants with history of HDP. This correlation was more evident for activin A and strongest with a history of preeclampsia. Our findings suggest that activin A may play an important role in blood pressure modulation in women with HDP who subsequently develop PPCM.

Immune Markers, Blood Pressure Severity, and Cardiac Remodeling 1 to 2 Years After Hypertensive Disorders of Pregnancy.

Background Cardiovascular dysfunction and hypertension can persist postpartum following hypertensive disorders of pregnancy (HDPs). This study hypothesized that activin A, proinflammatory markers and concentric remodeling by echo would be higher 1-2 years postpartum following HDP with persistent hypertension compared to HDP with normalized blood pressure (BP). We further hypothesized correlations between biomarkers with BP and echocardiographic indices. Methods and Results This study enrolled participants with HDPs but no prepregnancy hypertension followed 1 to 2 years after delivery. Activin A and inflammatory cytokines, BP, and echocardiograms were obtained. Biomarker concentrations and echocardiographic parameters were compared between HDP with and without persistent hypertension. Individuals with persistent hypertension at a mean of 1.6 years postpartum had significantly higher activin A concentrations (median[interquartile range 25-75] 230.6 [196.0-260.9] versus 175.3 pg/mL [164.3-188.4]; P<0.01), more concentric left ventricular concentric remodeling (relative wall thickness >0.42, 48% versus 7%; P<0.01), and worse peak left atrial strain (33.4% versus 39.3%; P<0.05) as compared with those whose BP normalized. Higher activin A and interleukin-6 concentrations correlated with higher systolic (activin A: r=0.43, P=0.01) and diastolic BP (activin A: r=0.58, P<0.01; interleukin-6: r=0.36; P<0.05), as well as greater left ventricular thickness (activin A and interventricular septal thickness: r=0.41, interleukin-6 and interventricular septal thickness: r=0.36; both P<0.05). Conclusions Individuals with HDPs and persistent hypertension had significantly higher activin A and greater concentric remodeling compared with those with HDPs and normalized BP at 1 to 2 years postpartum. Activin A was positively correlated with both BP and echocardiographic indices (left ventricular thickness), suggesting overlapping processes between persistent hypertension and cardiac remodeling.

Proinflammatory TH17 cytokine activation, disease severity and outcomes in peripartum cardiomyopathy.

BACKGROUND: Immune dysregulation is implicated in the development and clinical outcomes of peripartum cardiomyopathy (PPCM). METHODS AND RESULTS: 98 women with PPCM were enrolled and followed for 1 year postpartum (PP). LVEF was assessed at entry, 6-, and 12-months PP by echocardiography. Serum levels of soluble interleukin (IL)-2 receptor (sIL2R), IL-2, IL-4, IL-17, IL-22, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were measured by ELISA at entry. Cytokine levels were compared between women with PPCM by NYHA class. Outcomes including myocardial recovery and event-free survival were compared by cytokine tertiles. For cytokines found to impact survival outcomes, parameters indicative of disease severity including baseline LVEF, medications, and use of inotropic and mechanical support were analyzed. Levels of proinflammatory cytokines including IL-17, IL-22, and sIL2R, were elevated in higher NYHA classes at baseline. Subjects with higher IL-22 levels were more likely to require inotropic or mechanical support. Higher levels of TNF-α and IL-22 were associated with poorer event-free survival. Higher TNF-α levels were associated with lower mean LVEF at entry and 12 months. In contrast, higher levels of immune-regulatory cytokines such as IL-4 and IL-2 were associated with higher LVEF during follow up. CONCLUSION: Proinflammatory cytokines IL-22 and TNF-α were associated with adverse event-free survival. IL-17 and IL-22 were associated with more severe disease. In contrast, higher levels of IL-2 and IL-4 corresponded with higher subsequent LVEF. Increased production of TH17 type cytokines in PPCM correlated with worse disease and outcomes, while an increased immune-regulatory response seems to be protective.

Breastfeeding, Cellular Immune Activation, and Myocardial Recovery in Peripartum Cardiomyopathy.

The etiology of peripartum cardiomyopathy remains unknown. One hypothesis is that an increase in the 16-kDa form of prolactin is pathogenic and suggests that breastfeeding may worsen peripartum cardiomyopathy by increasing prolactin, while bromocriptine, which blocks prolactin release, may be therapeutic. An autoimmune etiology has also been proposed. The authors investigated the impact of breastfeeding on cellular immunity and myocardial recovery for women with peripartum cardiomyopathy in the IPAC (Investigations in Pregnancy Associated Cardiomyopathy) study. Women who breastfed had elevated prolactin, and prolactin levels correlated with elevations in CD8+ T cells. However, despite elevated prolactin and cytotoxic T cell subsets, myocardial recovery was not impaired in breastfeeding women.

Adrenergic Polymorphisms and Survival in African Americans With Heart Failure: Results From A-HeFT.

BACKGROUND: Polymorphisms in adrenergic signaling affect the molecular function of adrenergic receptors and related proteins. The ß1 adrenergic receptor (ADRB1) Arg389Gly, G-protein receptor kinase type 5 (GRK5) Gln41Leu, G-protein ß-3 subunit (GNB3) 825 C/T, and α2c deletion affect adrenergic tone, impact heart failure outcomes and differ in prevalence by ethnicity. Their combined effect within black cohorts remains unknown. METHODS AND RESULTS: We analyzed subjects from the African American Heart Failure Trial (A-HeFT) by assessing event-free survival, quality of life, and gene coinheritance. Significant coinheritance effects on survival included GRK5 Leu41 among subjects co-inheriting GNB3 825 C alleles (n = 166, 90.4% vs 69.0%, P < 0.001). By contrast, the impact of ADRB1 Arg389Arg genotype was magnified among subjects with GNB3 825 TT genotype (n = 181, 66.3% vs 85.7%, P = .002). The lack of the α2c deletion (ie, insertion) led to a greater impact of the ARG389Arg genotype (n = 289, 76.4% vs 86.1%, P = .007). CONCLUSIONS: Polymorphisms in adrenergic signaling affects outcomes in black subjects with heart failure. Coinheritance patterns in genetic variation may help determine heart failure survival.

Maternal Obesity Affects Cardiac Remodeling and Recovery in Women with Peripartum Cardiomyopathy.

OBJECTIVE: To examine the association between maternal obesity on left ventricular (LV) size and recovery in women with peripartum cardiomyopathy (PPCM). STUDY DESIGN: This was a prospective analysis of 100 women enrolled within 13 weeks of PPCM diagnosis and followed for a year in the Investigation of Pregnancy Associated Cardiomyopathy study. Adiposity was defined by standard body mass index (BMI) definitions for under/normal weight, overweight, and obesity. Demographic, clinical, and biomarker variables were compared across weight categories. OUTCOMES: LV end-diastolic diameter (LVEDD) and ejection fraction were measured at entry, 6, and 12 months postpartum. Multivariable regression models examined the relationship between adiposity, LV size, and leptin levels with cardiac recovery at 6 and 12 months postpartum. RESULTS: Obese and nonobese women had similar LV dysfunction at entry. Obese women had greater LV size and less LV recovery at 6 and 12 months postpartum. BMI was positively associated with leptin and ventricular diameter. Greater BMI at entry remained associated with less ventricular recovery at 6 months (p = 0.02) in adjusted race-stratified models. LVEDD at entry predicted lower ejection fraction at 6 months (p < 0.001) and similarly at 12 months. CONCLUSION: Obese women with PPCM had greater cardiac remodeling, higher leptin levels, and diminished cardiac recovery.

Association of Variants in BAG3 With Cardiomyopathy Outcomes in African American Individuals.

Importance: The prevalence of nonischemic dilated cardiomyopathy (DCM) is greater in individuals of African ancestry than in individuals of European ancestry. However, little is known about whether the difference in prevalence or outcomes is associated with functional genetic variants. Objective: We hypothesized that Bcl2-associated anthanogene 3 (BAG3) genetic variants were associated with outcomes in individuals of African ancestry with DCM. Design: This multicohort study of the BAG3 genotype in patients of African ancestry with dilated cardiomyopathy uses DNA obtained from African American individuals enrolled in 3 clinical studies: the Genetic Risk Assessment of African Americans With Heart Failure (GRAHF) study; the Intervention in Myocarditis and Acute Cardiomyopathy Trial-2 (IMAC-2) study; and the Genetic Risk Assessment of Cardiac Events (GRACE) study. Samples of DNA were also acquired from the left ventricular myocardium of patients of African ancestry who underwent heart transplant at the University of Colorado and University of Pittsburgh. Main Outcomes and Measures: The primary end points were the prevalence of BAG3 mutations in African American individuals and event-free survival in participants harboring functional BAG3 mutations. Results: Four BAG3 genetic variants were identified; these were expressed in 42 of 402 African American individuals (10.4%) with nonischemic heart failure and 9 of 107 African American individuals (8.4%) with ischemic heart failure but were not present in a reference population of European ancestry (P < .001). The variants included 2 nonsynonymous single-nucleotide variants; 1 three-nucleotide in-frame insertion; and 2 single-nucleotide variants that were linked in cis. The presence of BAG3 variants was associated with a nearly 2-fold (hazard ratio, 1.97 [95% CI, 1.19-3.24]; P = .01) increase in cardiac events in carriers compared with noncarriers. Transfection of transformed adult human ventricular myocytes with plasmids expressing the 4 variants demonstrated that each variant caused an increase in apoptosis and a decrease in autophagy when samples were subjected to the stress of hypoxia-reoxygenation. Conclusions and Relevance: This study demonstrates that genetic variants in BAG3 found almost exclusively in individuals of African ancestry were not causative of disease but were associated with a negative outcome in patients with a dilated cardiomyopathy through modulation of the function of BAG3. The results emphasize the importance of biological differences in causing phenotypic variance across diverse patient populations, the need to include diverse populations in genetic cohorts, and the importance of determining the pathogenicity of genetic variants.

Circulating T-Cell Subsets, Monocytes, and Natural Killer Cells in Peripartum Cardiomyopathy: Results From the Multicenter IPAC Study.

OBJECTIVE: The aim of this work was to evaluate the hypothesis that the distribution of circulating immune cell subsets, or their activation state, is significantly different between peripartum cardiomyopathy (PPCM) and healthy postpartum (HP) women. BACKGROUND: PPCM is a major cause of maternal morbidity and mortality, and an immune-mediated etiology has been hypothesized. Cellular immunity, altered in pregnancy and the peripartum period, has been proposed to play a role in PPCM pathogenesis. METHODS: The Investigation of Pregnancy-Associated Cardiomyopathy (IPAC) study enrolled 100 women presenting with a left ventricular ejection fraction of <0.45 within 2 months of delivery. Peripheral T-cell subsets, natural killer (NK) cells, and cellular activation markers were assessed by flow cytometry in PPCM women early (<6 wk), 2 months, and 6 months postpartum and compared with those of HP women and women with non-pregnancy-associated recent-onset cardiomyopathy (ROCM). RESULTS: Entry NK cell levels (CD3-CD56+CD16+; reported as % of CD3- cells) were significantly (P < .0003) reduced in PPCM (6.6 ± 4.9% of CD3- cells) compared to HP (11.9 ± 5%). Of T-cell subtypes, CD3+CD4-CD8-CD38+ cells differed significantly (P < .004) between PPCM (24.5 ± 12.5% of CD3+CD4-CD8- cells) and HP (12.5 ± 6.4%). PPCM patients demonstrated a rapid recovery of NK and CD3+CD4-CD8-CD38+ cell levels. However, black women had a delayed recovery of NK cells. A similar reduction of NK cells was observed in women with ROCM. CONCLUSIONS: Compared with HP control women, early postpartum PPCM women show significantly reduced NK cells, and higher CD3+CD4-CD8-CD38+ cells, which both normalize over time postpartum. The mechanistic role of NK cells and "double negative" (CD4-CD8-) T regulatory cells in PPCM requires further investigation.

Chemokine receptor patterns and right heart failure in mechanical circulatory support.

BACKGROUND: Right ventricular failure (RVF) complicates 9% to 44% of left ventricular assist device (LVAD) implants post-operatively. Current prediction scores perform only modestly in validation studies, and do not include immune markers. Chemokines are inflammatory signaling molecules with a fundamental role in cardiac physiology and stress adaptation. In this study we investigated chemokine receptor regulation in LVAD recipients who develop RVF. METHODS: Expression of chemokine receptor (CCR) genes 3 to 8 were examined in the peripheral blood of 111 LVAD patients, collected 24 hours before implant. RNA was isolated using a PAXgene protocol. Gene expression was assessed using a targeted microarray (RT2 Profiler PCR Array; Qiagen). Results were expressed as polymerase chain reaction (PCR) cycles to threshold and normalized to the average of 3 control genes, glyceraldehyde phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and ß2-microglobulin (B2M). Secondary outcomes studied were 1-year mortality and long-term RV failure (RVF-LT). RESULTS: CCR3, CCR4, CCR6, CCR7 and CCR8 were downregulated in LVAD recipients with RVF. Within this cohort of patients, CCR4, CCR7 and CCR8 were further downregulated in those who required RV mechanical support. In addition, under-expression of CCR3 to CCR8 was independently associated with an increased risk of mortality at 1 year, even after adjusting for RVF. CCR expression did not predict RVF-LT in our patient cohort. CONCLUSIONS: Pre-LVAD CCR downregulation is associated with RVF and increased mortality after implant. Inflammatory signatures may play a major role in prognostication in this patient population.

Relaxin-2 and Soluble Flt1 Levels in Peripartum Cardiomyopathy: Results of the Multicenter IPAC Study.

OBJECTIVES: This study explored the association of vascular hormones with myocardial recovery and clinical outcomes in peripartum cardiomyopathy (PPCM). BACKGROUND: PPCM is an uncommon disorder with unknown etiology. Angiogenic imbalance may contribute to its pathophysiology. METHODS: In 98 women with newly diagnosed PPCM enrolled in the Investigation in Pregnancy Associated Cardiomyopathy study, serum was obtained at baseline for analysis of relaxin-2, prolactin, soluble fms-like tyrosine kinase 1 (sFlt1), and vascular endothelial growth factor (VEGF). Left ventricular ejection fraction (LVEF) was assessed by echocardiography at baseline and 2, 6, and 12 months. RESULTS: Mean age was 30 ± 6 years, with a baseline of LVEF 0.35 ± 0.09. Relaxin-2, prolactin, and sFlt1 were elevated in women presenting early post-partum, but decreased rapidly and were correlated inversely with time from delivery to presentation. In tertile analysis, higher relaxin-2 was associated with smaller left ventricular systolic diameter (p = 0.006) and higher LVEF at 2 months (p = 0.01). This was particularly evident in women presenting soon after delivery (p = 0.02). No relationship was evident for myocardial recovery and prolactin, sFlt1 or VEGF levels. sFlt1 levels were higher in women with higher New York Heart Association functional class (p = 0.01) and adverse clinical events (p = 0.004). CONCLUSIONS: In women with newly diagnosed PPCM, higher relaxin-2 levels soon after delivery were associated with myocardial recovery at 2 months. In contrast, higher sFlt1 levels correlated with more severe symptoms and major adverse clinical events. Vascular mediators may contribute to the development of PPCM and influence subsequent myocardial recovery. (Investigation in Pregnancy Associate Cardiomyopathy [IPAC]; NCT01085955).

GNB3 C825T Polymorphism and Myocardial Recovery in Peripartum Cardiomyopathy: Results of the Multicenter Investigations of Pregnancy-Associated Cardiomyopathy Study.

BACKGROUND: Black women are at greater risk for peripartum cardiomyopathy (PPCM). The guanine nucleotide-binding proteins ß-3 subunit (GNB3) has a polymorphism C825T. The GNB3 TT genotype more prevalent in blacks is associated with poorer outcomes. We evaluated GNB3 genotype and myocardial recovery in PPCM. METHODS AND RESULTS: A total of 97 women with PPCM were enrolled and genotyped for the GNB3 T/C polymorphism. Left ventricular ejection fraction (LVEF) was assessed by echocardiography at entry, 6 and 12 months postpartum. LVEF over time in subjects with the GNB3 TT genotype was compared with those with the C allele overall and in black and white subsets. The cohort was 30% black, age 30+6, LVEF 0.34+0.10 at entry 31+25 days postpartum. The % GNB3 genotype for TT/CT/CC=23/41/36 and differed markedly by race (blacks=52/38/10 versus whites=10/44/46, P<0.001). In subjects with the TT genotype, LVEF at entry was lower (TT=0.31+0.09; CT+CC=0.35+0.09, P=0.054) and this difference increased at 6 (TT=0.45+0.15; CT+CC=0.53+0.08, P=0.002) and 12 months (TT=0.45+0.15; CT+CC=0.56+0.07, P<0.001.). The difference in LVEF at 12 months by genotype was most pronounced in blacks (12 months LVEF for GNB3 TT=0.39+0.16; versus CT+CC=0.53+0.09, P=0.02) but evident in whites (TT=0.50++0.11; CT+CC=0.56+0.06, P=0.04). CONCLUSIONS: The GNB3 TT genotype was associated with lower LVEF at 6 and 12 months in women with PPCM, and this was particularly evident in blacks. Racial differences in the prevalence and impact of GNB3 TT may contribute to poorer outcomes in black women with PPCM.

Shared Genetic Predisposition in Peripartum and Dilated Cardiomyopathies.

Background Peripartum cardiomyopathy shares some clinical features with idiopathic dilated cardiomyopathy, a disorder caused by mutations in more than 40 genes, including TTN, which encodes the sarcomere protein titin. Methods In 172 women with peripartum cardiomyopathy, we sequenced 43 genes with variants that have been associated with dilated cardiomyopathy. We compared the prevalence of different variant types (nonsense, frameshift, and splicing) in these women with the prevalence of such variants in persons with dilated cardiomyopathy and with population controls. Results We identified 26 distinct, rare truncating variants in eight genes among women with peripartum cardiomyopathy. The prevalence of truncating variants (26 in 172 [15%]) was significantly higher than that in a reference population of 60,706 persons (4.7%, P=1.3×10(-7)) but was similar to that in a cohort of patients with dilated cardiomyopathy (55 of 332 patients [17%], P=0.81). Two thirds of identified truncating variants were in TTN, as seen in 10% of the patients and in 1.4% of the reference population (P=2.7×10(-10)); almost all TTN variants were located in the titin A-band. Seven of the TTN truncating variants were previously reported in patients with idiopathic dilated cardiomyopathy. In a clinically well-characterized cohort of 83 women with peripartum cardiomyopathy, the presence of TTN truncating variants was significantly correlated with a lower ejection fraction at 1-year follow-up (P=0.005). Conclusions The distribution of truncating variants in a large series of women with peripartum cardiomyopathy was remarkably similar to that found in patients with idiopathic dilated cardiomyopathy. TTN truncating variants were the most prevalent genetic predisposition in each disorder.

G-protein beta-3 subunit genotype predicts enhanced benefit of fixed-dose isosorbide dinitrate and hydralazine: results of A-HeFT.

OBJECTIVES: The purpose of this study was to evaluate the influence of the guanine nucleotide-binding proteins (G-proteins), beta-3 subunit (GNB3) genotype on the effectiveness of a fixed-dose combination of isosorbide dinitrate and hydralazine (FDC I/H) in A-HeFT (African American Heart Failure Trial). BACKGROUND: GNB3 plays a role in alpha2-adrenergic signaling. A polymorphism (C825T) exists, and the T allele is linked to enhanced alpha-adrenergic tone and is more prevalent in African Americans. METHODS: A total of 350 subjects enrolled in the genetic substudy (GRAHF [Genetic Risk Assessment of Heart Failure in African Americans]) were genotyped for the C825T polymorphism. The impact of FDC I/H on a composite score (CS) that incorporated death, hospital stay for heart failure, and change in quality of life (QoL) and on event-free survival were assessed in GNB3 genotype subsets. RESULTS: The GRAHF cohort was 60% male, 25% ischemic, 97% New York Heart Association functional class III, age 57 ± 13 years, with a mean qualifying left ventricular ejection fraction of 0.24 ± 0.06. For GNB3 genotype, 184 subjects were TT (53%), 137 (39%) CT, and 29 (8%) were CC. In GNB3 TT subjects, FDC I/H improved the CS (FDC I/H = 0.50 ± 1.6; placebo = -0.11 ± 1.8, p = 0.02), QoL (FDC I/H = 0.69 ± 1.4; placebo = 0.24 ± 1.5, p = 0.04), and event-free survival (hazard ratio: 0.51, p = 0.047), but not in subjects with the C allele (for CS, FDC I/H = -0.05 ± 1.7; placebo = -0.09 ± 1.7, p = 0.87; for QoL, FDC I/H = 0.28 ± 1.5; placebo = 0.14 ± 1.5, p = 0.56; and for event-free survival, p = 0.35). CONCLUSIONS: The GNB3 TT genotype was associated with greater therapeutic effect of FDC I/H in A-HeFT. The role of the GNB3 genotype for targeting therapy with FDC I/H deserves further study.

Evidence for local relaxin ligand-receptor expression and function in arteries.

Relaxin is a 6 kDa protein hormone produced by the corpus luteum and secreted into the blood during pregnancy in rodents and humans. Growing evidence indicates that circulating relaxin causes vasodilatation and increases in arterial compliance, which may be among its most important actions during pregnancy. Here we investigated whether there is local expression and function of relaxin and relaxin receptor in arteries of nonpregnant females and males. Relaxin-1 and its major receptor, Lgr7, mRNA are expressed in thoracic aortas, small renal and mesenteric arteries from mice and rats of both sexes, as well as in small renal arteries from female tammar wallabies (an Australian marsupial). Using available antibodies for rat and mouse Lgr7 receptor and rat relaxin, we also identified protein expression in arteries. Small renal arteries isolated from relaxin-1 gene-deficient mice demonstrate enhanced myogenic reactivity and decreased passive compliance relative to wild-type (WT) and heterozygous mice. Taken together, these findings reveal an arterial-derived, relaxin ligand-receptor system that acts locally to regulate arterial function.

Evidence against the hypothesis that endothelial endothelin B receptor expression is regulated by relaxin and pregnancy.

The endothelial endothelin B (ET(B)) receptor subtype is critical for renal vasodilation induced by relaxin in nonpregnant rats and during pregnancy (the latter via endogenous circulating relaxin). Here we tested whether expression of vascular ET(B) receptor protein is regulated by relaxin. Small renal arteries were harvested from virgin and midterm pregnant rats as well as nonpregnant rats that were administered recombinant human relaxin (rhRLX) at 4 mug/h or vehicle for 5 d or 4-6 h. Small renal arteries dissected from additional virgin rats were incubated in vitro with rhRLX or vehicle for 3 h at 37 C. ET(B) expression was also evaluated in cultured human endothelial cells: aortic, coronary, umbilical vein, and dermal microvascular endothelial cells. Cells were incubated for 4, 8, or 24 h with rhRLX (5, 1, or 0.1 ng/ml) or vehicle. ET(B) protein expression in arteries and cells was evaluated by Western analysis. No regulation of ET(B) expression was observed in small renal arteries in any of the experimental protocols, nor was there an increase in the vasorelaxation response to ET-3 in small renal arteries incubated in vitro with rhRLX. rhRLX only sporadically altered ET(B) expression in human coronary artery endothelial cells and human umbilical vein endothelial cells at certain time points or doses, and no regulation was observed in human aortic endothelial cells or human dermal microvascular endothelial cells. These results suggest that regulation of ET(B) receptor protein has little or no role in relaxin stimulation of the endothelial ET(B)/nitric oxide vasodilatory pathway.