We have shown previously that 17β-estradiol (E2) increases left ventricular (LV) and cardiomyocyte hypertrophy following myocardial infarction (MI). However, E2 decreases hypertrophy in pressure overload models. We hypothesized that the effect of estrogen on cardiac hypertrophy was dependent on the type of hypertrophic stimulus.
Ovariectomized wild type female mice (n=192) were given vehicle or E2 treatment followed by coronary ligation (MI), transverse aortic constriction (TAC), or sham operation. Signaling pathway activation was studied at 3, 24, and 48 hours while echocardiography and hemodynamic studies were performed at 14 days.
MI induced early but transient activation of p38 and p42/44 MAPK pathways, whereas TAC induced sustained activation of both pathways. E2 had no effect on these pathways, but increased Stat3 activation following MI while decreasing Stat3 activation following TAC. MI caused LV dilation, and decreased fractional shortening (FS), that were unaltered by E2. TAC caused LV dilation, reduced FS, and increased LV mass, but in this model, E2 improved these parameters. Following MI, E2 led to increases in myocyte cross-sectional area, atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC) gene expression, but E2 diminished TAC-induced increases ANP and β-MHC gene expression.
These data demonstrate that the effects of E2 on LV and myocyte remodeling depend on the nature of the hypertrophic stimulus. The opposing influence of E2 on hypertrophy in these models may, in part, result from differential effects of E2 on Stat3 activation. Further work will be necessary to explore this and other potential mechanisms by which estrogen affects hypertrophy in these models.
Estrogen; LV Remodeling; Myocyte Hypertrophy; Myocardial Infarction; Pressure Overload; Signal Transducer and Activator of Transcription
The proliferation of vascular smooth muscle cells (VSMC) plays a crucial role in vascular diseases such as atherosclerosis and restenosis after percutaneous coronary intervention. Many studies have shown that estrogen inhibits VSMC proliferation in response to vascular injury in the mouse carotid injury model. However, the mechanisms that mediate these effects remain unclear. We here investigated the mechanisms by which estrogen inhibits VSMC proliferation.
Approach and Results
We established a novel transgenic mouse line, referred to as the disrupting peptide mice (DPM), in which rapid estrogen receptor (ER)-mediated signaling is abolished by overexpression of a peptide that prevents the ER from forming a signaling complex necessary for rapid signaling. Carotid artery VSMC from DPM or littermate wild type (WT) female mice were obtained by the explant method. In VSMC derived from WT mice, estrogen significantly inhibited VSMC proliferation. Phosphorylation levels of Akt and ERK induced by PDGF were significantly inhibited by estrogen pretreatment. Estrogen enhanced complex formation between ERα and protein phosphatase 2 (PP2) A, and enhanced PP2A activity. The blockade of PP2A activity abolished the estrogen-induced anti-proliferative effect on VSMC. In contrast, none of these effects of estrogen observed in the WT VSMC were observed in VSMC derived from DPM. These results support that rapid non-nuclear ER signaling is required for estrogen-induced inhibition of VSMC proliferation, and further that PP2A activation by estrogen mediates estrogen-induced anti-proliferative effects.
These findings support that PP2A activation via rapid non-nuclear ER signaling may be a novel target for therapeutic approaches to inhibit VSMC proliferation, which plays a central role in atherosclerosis and restenosis.
cardiovascular diseases; hormones; molecular biology; signal transduction
Clinical trial and epidemiological data support that the cardiovascular effects of estrogen are complex, including a mixture of both potentially beneficial and harmful effects. In animal models, estrogen protects females from vascular injury and inhibits atherosclerosis. These effects are mediated by estrogen receptors (ERs), which when bound to estrogen can bind to DNA to directly regulate transcription. ERs can also activate several cellular kinases by inducing a “rapid” non-nuclear signaling cascade. However, the biologic significance of this rapid signaling pathway has been unclear.
Methods and Results
Here, we develop a novel transgenic mouse in which rapid signaling is blocked by over-expression of a peptide that prevents ERs from interacting with the scaffold protein, striatin (the Disrupting Peptide Mouse, DPM). Microarray analysis of ex vivo-treated mouse aortas demonstrates that rapid ER signaling plays an important role in estrogen-mediated gene regulatory responses. Disruption of ER-striatin interactions also eliminates the ability of estrogen to stimulate cultured endothelial cell migration and to inhibit cultured vascular smooth muscle cell growth. The importance of these findings is underscored by in vivo experiments demonstrating loss of estrogen-mediated protection against vascular injury in the DPM mouse following carotid artery wire injury.
Taken together, these results support that rapid, non-nuclear ER signaling contributes to the transcriptional regulatory functions of ER, and is essential for many of the vasoprotective effects of estrogen. These findings also identify the rapid ER signaling pathway as a potential target for the development of novel therapeutic agents.
cardiovascular diseases; hormones; molecular biology; signal transduction
Estradiol (E2) regulates gene transcription by activating estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Many of the genes regulated by E2 via ERs are repressed, yet the molecular mechanisms that mediate E2-induced gene repression are currently unknown. We hypothesized that E2, acting through ERs, regulates expression of microRNAs (miRs) leading to repression of expression of specific target genes.
Methods and Results
Here, we report that E2 significantly up-regulates the expression of 26 miRs and down-regulates the expression of 6 miRs in mouse aorta. E2 mediated up-regulation of one of these miRs, miR-203, was chosen for further study. In cultured vascular smooth muscle cells (VSMC), E2-mediated up-regulation of miR-203 is mediated by ERα (but not ERβ) via transcriptional up-regulation of the primary miR. We demonstrate that the transcription factors Zeb-1 and AP-1 play critical roles in mediating E2-induced up-regulation of miR-203 transcription. We show further that miR-203 mediates E2-induced repression of Abl1 and p63 protein abundance in VSMC. Finally, knocking-down miR-203 abolishes E2-mediated inhibition of VSMC proliferation, and over-expression of miR-203 inhibits cultured VSMC proliferation, but not vascular endothelial cell proliferation.
Our findings demonstrate that E2 regulates expression of miRs in the vasculature, and support that ER-dependent induction of miRs is a mechanism for E2-mediated gene repression. Furthermore, our findings demonstrate that miR-203 contributes to E2-induced inhibition of VSMC proliferation and highlight the potential of miR-203 as a therapeutic agent in the treatment of proliferative cardiovascular diseases.
estrogen; gene regulation; microRNA; muscle, smooth; proliferation
Hypertensive heart disease causes significant mortality in older patients, yet there is an incomplete understanding of molecular mechanisms that regulate age-dependent hypertensive left ventricular hypertrophy (LVH). Therefore, we tested the hypothesis that the cGMP-dependent protein kinase G I alpha (PKGIα) attenuates hypertensive LVH by evaluating the cardiac phenotype in mice with selective mutations of the PKGIα leucine zipper domain. These leucine zipper mutant (LZM) mice develop basal hypertension. Compared with wild-type controls, 8-month-old adult LZM mice developed increased left ventricular end-diastolic pressure but without frank LVH. In advanced age (15 months), the LZM mice developed overt pathological LVH. These findings reveal a role of PKGIα in normally attenuating hypertensive LVH. Therefore, mutation of the PKGIα LZ domain produces a clinically relevant model for hypertensive heart disease of aging.
Left ventricular hypertrophy; Hypertension; Protein kinase G.
Unique innate immunity-linked γδT cells have been seen in early human artery lesions, but their role in lesion development has received little attention. Here we investigated whether γδT cells modulate atherogenesis in apolipoprotein E-deficient (ApoE KO) mice. We found that γδT cell numbers were markedly increased in the proximal aorta of ApoE-deficient vs. wild-type mice during early atherogenesis, particularly in the aortic root and arch, where they comprised most of the T cells and lesion progression is most rapid. γδT cells infiltrated intimal lesions in ApoE KO mice, but only the adventitia in wild-type mice, and were more prevalent than CD4+ T cells in early nascent lesions, as evaluated by en face confocal microscopy. These aortic γδT cells produced IL-17, but not IFN-γ, analyzed by ex vivo FACS. Furthermore, aortic arch lipid accumulation correlated strongly with abundance of IL-17-expressing splenic γδT cells in individual ApoE KO mice. To investigate the role of these γδT cells in early atherogenesis, we analyzed ApoE/γδT double knockout (DKO) compared to ApoE KO mice. We observed reduced early intimal lipid accumulation at sites of nascent lesion formation, both in chow-fed (by 40%) and Western diet-fed (by 44%) ApoE/γδT DKO mice. In addition, circulating neutrophils were drastically reduced in these DKO mice on Western diet, while expansion of inflammatory monocytes and splenic Th1 or Th17 lymphocytes was not affected. These data reveal, for the first time, a pathogenic role of γδT cells in early atherogenesis in ApoE KO mice, by mechanisms likely to involve their IL-17 production and induction of neutrophilia. Targeting γδT cells thus might offer therapeutic benefit in atherosclerosis or other inflammatory vascular diseases.
Right ventricular (RV) failure is a major cause of mortality worldwide and is often a consequence
of RV pressure overload (RVPO). Endoglin is a coreceptor for the profibrogenic cytokine,
transforming growth factor beta 1 (TGF‐β1). TGF‐β1 signaling by the
canonical transient receptor protein channel 6 (TRPC‐6) was recently reported to stimulate
calcineurin‐mediated myofibroblast transformation, a critical component of cardiac fibrosis.
We hypothesized that reduced activity of the TGF‐β1 coreceptor, endoglin, limits RV
calcineurin expression and improves survival in RVPO.
Methods and Results
We first demonstrate that endoglin is required for TGF‐β1‐mediated
calcineurin/TRPC‐6 expression and up‐regulation of alpha‐smooth muscle
antigen (α‐SMA), a marker of myofibroblast transformation, in human RV fibroblasts.
Using endoglin haploinsufficient mice (Eng+/−) we show that reduced
endoglin activity preserves RV function, limits RV fibrosis, and attenuates activation of the
calcineurin/TRPC‐6/α‐SMA pathway in a model of
angio‐obliterative pulmonary hypertension. Next, using Eng+/−
mice or a neutralizing antibody (Ab) against endoglin (N‐Eng) in wild‐type mice, we
show that reduced endoglin activity improves survival and attenuates RV fibrosis in models of RVPO
induced by pulmonary artery constriction. To explore the utility of targeting endoglin, we observed
a reversal of RV fibrosis and calcineurin levels in wild‐type mice treated with a
N‐Eng Ab, compared to an immunoglobulin G control.
These data establish endoglin as a regulator of TGF‐β1 signaling by calcineurin and
TRPC‐6 in the RV and identify it as a potential therapeutic target to limit RV fibrosis and
improve survival in RVPO, a common cause of death in cardiac and pulmonary disease.
heart failure; pulmonary hypertension; fibrosis; right ventricle; cardiac remodeling
Vein graft failure rates due to adverse graft remodeling remain high with no effective therapy. The mineralocorticoid receptor (MR) plays a role in pathologic arterial remodeling. We have recently demonstrated that MR is upregulated in venous tissues after grafting and hypothesized that MR inhibition would reduce vein graft remodeling.
Reverse transcription polymerase chain reaction and immunoblotting were used to examine the expression of MR and other components of the renin-angiotensin-aldosterone system in human vein and primary human saphenous vein smooth muscle cells (HSVSMC). Adenoviral reporter gene assays were used to explore MR transcriptional activity in HSVSMC. The effect of MR inhibition on vein graft remodeling in vivo was characterized in a mouse vein graft model.
Messenger RNAs encoding MR, 11-β-hydroxysteroid dehydrogenase 2 (11bHSD2), angiotensin type-1 receptor, and the angiotensin converting enzyme are expressed in whole human vein and in HSVSMC. MR and 11βHSD2 protein expression is confirmed and MR-dependent transcriptional regulation is demonstrated at physiologic aldosterone concentrations in HSVSMC. Treatment of mice with the MR antagonist spironolactone, at doses that do not lower blood pressure (20 mg/kg/day), reduces maximal vein graft intima-media thickness by 68%, with an associated reduction in graft inflammatory cell infiltration and fibrosis.
The MR is expressed in human venous tissue and cells and modulates gene expression in HSVSMC in response to physiologic aldosterone concentrations. In vivo, MR inhibition reduces vein graft thickening and inflammation. These preclinical data support the potential to use MR antagonists as novel treatments to preserve vein graft patency.
Inhibition of cGMP-specific phosphodiesterase 5 (PDE5) ameliorates pathological cardiac remodeling and has been gaining attention as a potential therapy for heart failure. Despite promising results in males, the efficacy of the PDE5 inhibitor sildenafil in female cardiac pathologies has not been determined and might be affected by estrogen levels, given the hormone’s involvement in cGMP synthesis. Here, we determined that the heart-protective effect of sildenafil in female mice depends on the presence of estrogen via a mechanism that involves myocyte eNOS–dependent cGMP synthesis and the cGMP-dependent protein kinase Iα (PKGIα). Sildenafil treatment failed to exert antiremodeling properties in female pathological hearts from Gαq-overexpressing or pressure-overloaded mice after ovary removal; however, estrogen replacement restored the effectiveness of sildenafil in these animals. In females, sildenafil-elicited myocardial PKG activity required estrogen, which stimulated tonic cardiomyocyte cGMP synthesis via an eNOS/soluble guanylate cyclase pathway. In contrast, eNOS activation, cGMP synthesis, and sildenafil efficacy were not estrogen dependent in male hearts. Estrogen and sildenafil had no impact on pressure-overloaded hearts from animals expressing dysfunctional PKGIα, indicating that PKGIα mediates antiremodeling effects. These results support the importance of sex differences in the use of PDE5 inhibitors for treating heart disease and the critical role of estrogen status when these agents are used in females.
Estrogen receptors are best known as ligand-activated transcription factors that regulate vascular cell gene expression. For many years now, a rapid signaling pathway mediated by cell membrane–associated estrogen receptors also has been recognized, but the physiological relevance of this pathway has remained unclear. In this issue of the JCI, Chambliss et al. provide new data to indicate that activation of non-nuclear estrogen receptor signaling regulates processes central to cardiovascular health and disease. These investigators show that an estrogen-dendrimer conjugate (EDC), which activates estrogen receptors but remains non-nuclear, stimulates vascular EC migration in vitro and protects against vascular injury in vivo. They show further that the vascular benefits of EDC in vivo occur selectively in the vasculature, without stimulating the uterus or enhancing growth of breast cancer xenografts. Taken together, these findings indicate that activation of non-nuclear estrogen receptor signaling regulates vascular events of physiological relevance and suggest that translation of these findings into clinically relevant therapeutic interventions is a logical next goal.
Right ventricular (RV) failure is a major cause of mortality in acute or chronic lung disease and left heart failure. The objective of this study was to demonstrate a percutaneous approach to study biventricular hemodynamics in murine models of primary and secondary RV pressure overload (RVPO) and further explore biventricular expression of two key proteins that regulate cardiac remodeling: calcineurin and transforming growth factor beta 1 (TGFβ1).
Adult, male mice underwent constriction of the pulmonary artery or thoracic aorta as models of primary and secondary RVPO, respectively. Conductance catheterization was performed followed by tissue analysis for changes in myocyte hypertrophy and fibrosis.
Both primary and secondary RVPO decreased biventricular stroke work however RV instantaneous peak pressure (dP/dtmax) and end-systolic elastance (Ees) were preserved in both groups compared to controls. In contrast, left ventricular (LV) dP/dtmax and LV-Ees were unchanged by primary, but reduced in the secondary RVPO group. The ratio of RV:LV ventriculo-arterial coupling was increased in primary and reduced in secondary RVPO. Primary and secondary RVPO increased RV mass, while LV mass decreased in primary and increased in the secondary RVPO groups. RV fibrosis and hypertrophy were increased in both groups, while LV fibrosis and hypertrophy were increased in secondary RVPO only. RV calcineurin expression was increased in both groups, while LV expression increased in secondary RVPO only. Biventricular TGFβ1 expression was increased in both groups.
These data identify distinct effects of primary and secondary RVPO on biventricular structure, function, and expression of key remodeling pathways.
Although cigarette smoking is a risk factor for heart failure (HF), smokers with HF have lower mortality rates during/following hospitalization compared to nonsmokers. We examined vascular endothelial function in chronic smokers and nonsmokers with HF as it relates to this smoker’s paradox.
Brachial artery flow mediated dilation (FMD), a measure of conduit vessel endothelial function, was measured in 33 smoking and nonsmoking patients with HF versus controls. In addition, soluble endoglin (sEng), a circulating mediator of endothelial function, was measured in a separate group of 36 smoking and non-smoking patients with HF versus controls.
FMD was significantly lower in smokers without HF compared the nonsmokers without HF (p<0.05). FMD was significantly higher in smokers with HF vs. non-smokers with HF (p<0.05) and did not differ from values seen in nonsmokers without HF (p>0.05). There were no differences in sEng between smokers and nonsmokers without HF (p>0.05). sEng was lower in smokers with HF vs. non-smokers with HF (p < 0.05) and did not differ from values seen in nonsmokers without HF (p>0.05).
Smokers with HF had higher brachial FMD and lower sEng than nonsmokers with HF and values were comparable to nonsmokers without HF. These findings offer novel insight into the smoker’s paradox and suggest that improved short-term outcome in patients hospitalized with HF may in part be mediated by preservation of vascular endothelial function in this setting.
Cyclic GMP (cGMP) signaling attenuates cardiac remodeling, but it is unclear which cGMP effectors mediate these effects and thus might serve as novel therapeutic targets. Therefore, we tested whether the cGMP downstream effector, cGMP-dependent protein kinase G Iα (PKGIα), attenuates pressure overload–induced remodeling in vivo.
Methods and Results
The effect of transaortic constriction (TAC)–induced left ventricular (LV) pressure overload was examined in mice with selective mutations in the PKGIα leucine zipper interaction domain. Compared with wild-type littermate controls, in response to TAC, these Leucine Zipper Mutant (LZM) mice developed significant LV systolic and diastolic dysfunction by 48 hours (n=6 WT sham, 6 WT TAC, 5 LZM sham, 9 LZM TAC). In response to 7-day TAC, the LZM mice developed increased pathologic hypertrophy compared with controls (n=5 WT sham, 4 LZM sham, 8 WT TAC, 11 LZM TAC). In WT mice, but not in LZM mice, phosphodiesterase 5 (PDE5) inhibition with sildenafil (Sil) significantly inhibited TAC-induced cardiac hypertrophy and LV systolic dysfunction in WT mice, but this was abolished in the LZM mice (n=3 WT sham, 4 LZM sham, 3 WT TAC vehicle, 6 LZM TAC vehicle, 4 WT TAC Sil, 6 LZM TAC Sil). And in response to prolonged, 21-day TAC (n=8 WT sham, 7 LZM sham, 21 WT TAC, 15 LZM TAC), the LZM mice developed markedly accelerated mortality and congestive heart failure. TAC induced activation of JNK, which inhibits cardiac remodeling in vivo, in WT, but not in LZM, hearts, identifying a novel signaling pathway activated by PKGIα in the heart in response to LV pressure overload.
These findings reveal direct roles for PKGIα in attenuating pressure overload–induced remodeling in vivo and as a required effector for the cardioprotective effects of sildenafil.
heart failure; nitric oxide; protein kinase G; remodeling heart failure; signal transduction
Background. Cardiovascular disease is the leading cause of premature mortality in autosomal dominant polycystic kidney disease (ADPKD). We examined peripheral augmentation index (AIx) as a measure of systemic vascular function and circulating markers of vascular inflammation in patients with ADPKD.
Methods. Fifty-two ADPKD patients with hypertension and estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2, 50 ADPKD patients with hypertension and eGFR ≥60 mL/min/1.73 m2, 42 normotensive ADPKD patients with eGFR ≥60 mL/min/1.73 m2 and 51 normotensive healthy controls were enrolled in this study. AIx was measured from peripheral artery tone recordings using finger plethysmography. Serum levels of soluble intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1, P-selectin, E-selectin, soluble Fas (sFas) and Fas ligand (FasL) were measured as markers of vascular inflammation.
Results. AIx was higher in all three patient groups with ADPKD compared to healthy controls (P < 0.05). AIx was similar between the normotensive ADPKD patients with eGFR ≥60 mL/min/1.73 m2 and hypertensive ADPKD patients with eGFR <60 mL/min/1.73 m2 (P > 0.05). ICAM, P-selectin, E-selectin and sFas were higher and FasL lower in all ADPKD groups compared to controls (P < 0.05). ICAM, P-selectin and E-selectin were similar between the normotensive ADPKD patients with eGFR ≥60 mL/min/1.73 m2 and hypertensive ADPKD patients with eGFR < 60 mL/min/1.73 m2 (P > 0.05). According to multiple regression analysis, predictors of AIx in ADPKD included age, height, heart rate and mean arterial pressure (P < 0.05). Vascular inflammatory markers were not predictors of AIx in ADPKD.
Conclusions. Systemic vascular dysfunction, manifesting as an increase in AIx and vascular inflammation is evident in young normotensive ADPKD patients with preserved renal function. Vascular inflammation is not associated with elevated AIx in ADPKD.
hypertension; inflammation; kidney disease; vascular function
In addition to the classical nuclear estrogen receptor, the expression of non-nuclear estrogen receptors localized to the cell surface membrane (mER) has recently been demonstrated. Estrogen and its receptors have been implicated in the development or progression of numerous neurodegenerative disorders. Furthermore, the pathogenesis of these diseases has been associated with disturbances of two key cellular programs: apoptosis and autophagy. An excess of apoptosis or a defect in autophagy has been implicated in neurodegeneration. The aim of this study was to clarify the role of ER in determining neuronal cell fate and the possible implication of these receptors in regulating either apoptosis or autophagy. The human neuronal cell line SH-SY5Y and mouse neuronal cells in primary culture were thus exposed to chronic minimal peroxide treatment (CMP), a form of subcytotoxic minimal chronic stress previously that mimics multiple aspects of long-term cell stress and represents a limited molecular proxy for neurodegenerative processes. We actually found that either E2 or E2-bovine serum albumin construct (E2BSA, i.e. a non-permeant form of E2) was capable of modulating intracellular cell signals and regulating cell survival and death. In particular, under CMP, the up-regulation of mERα, but not mERβ, was associated with functional signals (ERK phosphorylation and p38 dephosphorylation) compatible with autophagic cytoprotection triggering and leading to cell survival. The mERα trafficking appeared to be independent of the microfilament system cytoskeletal network but was seemingly associated with microtubular apparatus network, i.e., to MAP2 molecular chaperone. Importantly, antioxidant treatments, administration of siRNA to ERα, or the presence of antagonist of ERα hindered these events. These results support that the surface expression of mERα plays a pivotal role in determining cell fate, and that ligand-induced activation of mER signalling exerts a powerful cell-survival signal. These results shed new light on the pathogenetic mechanisms leading to neuronal cell degeneration.
Progressive left ventricular (LV) dysfunction induces expression of the cytokine transforming growth factor-beta (TGFb1). Endoglin (CD105) is a TGFb1 co-receptor that is released into the circulation as soluble endoglin (sEng). The objective of this study was to assess serum levels of sEng in heart failure and to identify the predictive value of sEng for detecting elevated left ventricular end-diastolic pressures (LVEDP). We measured sEng levels in 82 consecutive patients with suspected LV dysfunction referred for determination of left heart filling pressures by cardiac catheterization. Among these subjects sEng levels correlated with LVEDP (R=0.689; p<0.0001) irrespective of LV ejection fraction (LVEF). Using a receiving operative characteristic (ROC) curve, sEng levels predicted an LVEDP≥16mmHg with an area-under-the-curve (AUC) of 0.85, exceeding measured AUCs for both atrial- and brain-natriuretic peptide, currently used biomarkers for heart failure diagnosis (ANP:0.68; BNP:0.65, p<0.01 vs sEng respectively). In 10 subjects receiving medical therapy for heart failure guided by invasive hemodynamic monitoring, decreased pulmonary capillary wedge pressure was associated with a reduced sEng level (R=0.75, p=0.008). Finally, compared to 25 healthy controls, sEng levels were elevated in subjects with suspected LV dysfunction (3589±588 vs 4257±966 pg/mL, respectively, p<0.005) and correlated directly with New York Heart Association class (NYHA; R=0.501, p<0.001). In conclusion, circulating levels of sEng are elevated in patients with increased LVEDP and NYHA class, irrespective of LVEF. Soluble endoglin levels also decrease in association with reduced cardiac filling pressure after diuresis. These findings identify circulating sEng as a sensitive measure of elevated left heart filling pressure.
heart failure; cardiac filling pressure; soluble receptors
Human studies of therapeutic angiogenesis, stem-cell, and progenitor-cell therapy have failed to demonstrate consistent clinical benefit. Recent studies have shown that heparin increases circulating levels of anti-angiogenic peptides. Given the widely prevalent use of heparin in percutaneous and surgical procedures including those performed as part of studies examining the benefit of therapeutic angiogenesis and cell-based therapy, we compared the effects of unfractionated heparin (UFH) on angiogenic peptides with those of bivalirudin, a relatively newer anticoagulant whose effects on angiogenic peptides have not been studied.
We measured soluble fms-like tyrosine kinase-1 (sFLT1), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), and soluble Endoglin (sEng) serum levels by enzyme linked immunosorbent assays (ELISA) in 16 patients undergoing elective percutaneous coronary intervention. Compared to baseline values, sFLT1 and PlGF levels increased by 2629±313% and 253±54%, respectively, within 30 minutes of UFH therapy (p<0.01 for both; n = 8). VEGF levels decreased by 93.2±5% in patients treated with UFH (p<0.01 versus baseline). No change in sEng levels were observed after UFH therapy. No changes in sFLT1, PlGF, VEGF, or sEng levels were observed in any patients receiving bivalirudin (n = 8). To further explore the direct effect of anticoagulation on circulating angiogenic peptides, adult, male wild-type mice received venous injections of clinically dosed UFH or bivalirudin. Compared to saline controls, sFLT1 and PlGF levels increased by >500% (p<0.01, for both) and VEGF levels increased by 221±101% (p<0.05) 30 minutes after UFH treatment. Bivalirudin had no effect on peptide levels. To study the cellular origin of peptides after anticoagulant therapy, human coronary endothelial cells were treated with UFH and demonstrated increased sFLT1 and PlGF levels (ANOVA p<0.01 for both) with reduced VEGF levels (ANOVA p<0.05). Bivalirudin had no effect on peptide levels in vitro.
Circulating levels of sFLT1, PlGF, and VEGF are significantly altered by UFH, while bivalirudin therapy has no effect. These findings may have significant implications for clinical studies of therapeutic angiogenesis, stem-cell and progenitor-cell therapy.
Almost one-third of patients with hypertrophic cardiomyopathy (HC) have an abnormal blood pressure response (ABPR) to exercise and this is associated with a greater risk of sudden cardiac death (SCD). In this study, we examined the association between steady (mean arterial pressure) and pulsatile (pulse pressure) blood pressure components as they relate to ABPR in patients with HC (n = 70). All patients completed a standard Bruce protocol during symptom limited stress testing with concurrent hemodynamic measurements. PP was significantly higher in HC patients with ABPR (n = 19) compared to HC patients with a normal BP response to exercise (p<0.05). According to binary logistic regression, resting PP was a significant predictor of ABPR in patients with HC (p<0.05). MAP was not significantly different between groups, nor was it a predictor of ABPR in HC. Those within the highest tertile of resting PP were 4.8 times more likely to have an ABPR compared to those within the lowest PP tertile (95% CI = 1.24 – 18.2, p<0.05). In conclusion, elevations in resting PP may identify HC individuals at higher risk for having an ABPR during exercise.
Striatin, a putative protein phosphatase 2A (PP2A) B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM), which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit) heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3) protein, the mammalian Mps one binder (MOB) homolog, Mob3/phocein, the mammalian sterile 20-like (Mst) kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases.
To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3.
Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via residues lying between striatin's calmodulin-binding and WD-domains and recruits the PP2A A/C heterodimer to its coiled-coil/oligomerization domain. Residues outside the previously reported coiled-coil domain of striatin are necessary for its oligomerization. Striatin-associated PP2A is critical for Mst3 dephosphorylation and inactivation. Upon inhibition of PP2A, Mst3 activation appears to involve autophosphorylation of multiple activation loop phosphorylation sites. Mob3 can associate with striatin sequences C-terminal to the Mst3 binding site but also with sequences proximal to striatin-associated PP2A, consistent with a possible role for Mob 3 in the regulation of Mst3 by PP2A.
Although typically derived from the contour of arterial pressure waveform, augmentation index (AIx) may also be derived from the digital pulse volume waveform using finger plethysmography (peripheral arterial tonometry, PAT). Little is known regarding the physiologic correlates of AIx derived from PAT. In this study, we investigated the relation of PAT-AIx with measures of ventricular-vascular coupling.
Pulse volume waves were measured via PAT and used to derive AIx. Using 2-dimensional echocardiography, effective arterial elastance index (EaI) was estimated as end systolic pressure / stroke volume index. Left ventricular (LV) end-systolic elastance index (ELVI) was calculated as end systolic pressure / end systolic volume index. Ventricular-vascular coupling ratio was defined as EaI/ELVI.
Given the bi-directional nature of ventricular-vascular uncoupling as measured by echocardiography, patients were separated into 3 groups: low EaI/ELVI (<0.6, n = 21), optimal EaI/ELVI (mean 0.6–1.2, n = 16) and high EaI/ELVI (>1.2, n = 10). Adjusting for potential confounders (age, mean arterial pressure, height, and heart rate) patients with optimal EaI/ELVI had lower AIx (1±4%, p<0.05) compared to those with low EaI/ELVI (13±4%) and high EaI/ELVI (19±5%).
Abnormal ventricular-vascular coupling, arising from either increased effective arterial elastance or increased ventricular elastance, is associated with increased AIx as measured by PAT. Additional research is needed to examine other vascular correlates of PAT-AIx.
augmentation index; effective arterial elastance; Left ventricular end-systolic elastance
Background. Augmentation index (AIx) is traditionally obtained from pressure waveforms via arterial applanation tonometry. We sought to evaluate the association between
AIx obtained from peripheral arterial tonometry (PAT) with cardiovascular risk factors (CRF) and coronary artery disease (CAD).
Methods. 186 patients were enrolled in the study. The presence or absence of CRFs and CAD was assessed in each subject. AIx was calculated by an automated algorithm averaging pulse wave amplitude data obtained via PAT. Central blood pressures were assessed in a subset of patients undergoing clinically indicated cardiac catheterization. Results. An association was observed between AIx and age, heart rate, systolic blood pressure, mean arterial pressure, pulse pressure, body weight and body mass index. AIx was significantly lower in patients with <3
CRFs compared to those with >5 CRFs ( P = .02). CAD+ patients had significantly higher AIx compared to CAD− patients ( P = .008). Area under the ROC curve was 0.604 (P < .01). In patients undergoing cardiac catheterization, after adjusting for age, height and heart rate, AIx was a significant predictor of aortic systolic and pulse pressures (P < .05) Conclusion. AIx derived from PAT correlates with cardiac risk factors and CAD. It may be a useful measure of assessing overall risk for coronary artery disease.
Coronary artery disease (CAD) risk is not fully revealed by traditional risk factors. Identification of a simple, non-invasive tool that allows for detection of high risk CAD patients and can be applied in large population/clinical settings would prove valuable.
In this study, finger peripheral arterial tonometry (PAT) was used to measure pulse wave amplitude (PWA) during reactive hyperemia (RH) and taken as a measure of microvascular endothelial function in 42 men with stable CAD and well-controlled LDL-cholesterol levels. Plasma levels of high sensitivity C-reactive protein (hs-CRP) and lipoprotein-associated phospholipase A2 (Lp-PLA2) were measured and used to re-classify men into high-risk (elevated hs-CRP and Lp-PLA2), moderate risk (either elevated hs-CRP or Lp-PLA2) or low risk (low hs-CRP and Lp-PLA2) groups.
PWA-RH was significantly lower in the high risk group (1.3±0.04) compared to the moderate risk (1.6±0.07, p<0.05) and low risk (2.0±0.1, p<0.05) groups. According to binary logistic regression, PWA-RH was a significant predictor of high-risk status amongst men with CAD (p<0.05).
Measurement of peripheral microvascular endothelial function with PAT may be able to distinguish high-risk from moderate and low-risk men with stable CAD and well-controlled LDL-cholesterol levels and thus aid in residual risk stratification in this at risk cohort.
endothelial function; inflammation; cardiovascular
Patients with hypertrophic cardiomyopathy (HC) have coronary microvascular dysfunction which is an independent predictor of adverse left ventricular remodeling, systolic dysfunction, and mortality in these patients. Whether these defects in vasomotor function are localized to the coronary arteries or whether systemic vasomotor dysfunction is present in HC patients has not yet been adequatley examined. We tested the hypothesis that patients with HC (n = 46) have altered peripheral vascular endothelial function. Subjects without coronary artery disease (CAD− n = 46) and subjects with CAD (CAD+, n = 46), served as negative and positive controls, respectively. Conduit artery endothelium-dependent vasomotion was assessed with ultrasound by measuring flow-mediated dilation (FMD) of the brachial artery. FMD was lower in HC patients compared to CAD− (p<0.05) but was similar to CAD+ patients (p=NS). In conclusion, vasomotor dysfunction in HC is not restricted to the coronary vasculature. Patients with HC have impaired peripheral conduit vessel endothelial function and the magnitude of impairment is similar to that seen in older patients with advanced CAD.
cardiomyopathy; flow mediated dilation; vascular function