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Aims: The aim of this study was to investigate whether galectin-3 (Gal-3) is associated with myocardial histological and molecular parameters related to fibrosis and with the circulating biomarkers of the extracellular generation of mature fibril-forming collagen types I (C-terminal propeptide of procollagen type I, PICP) and III (N-terminal propeptide of procollagen type III, PIIINP) in two independent studies of hypertensive patients with heart failure (HF)., Methods and Results: Endomyocardial biopsies and blood samples from 39 HF patients (invasive study), and blood samples from 220 HF patients (non-invasive study) were analysed. Necropsies (n = 7) and blood samples (n = 20) from healthy subjects were used as controls. In the invasive study myocardial mRNA and protein expression of Gal-3 and collagen types I and III, plasma Gal-3 and serum PICP and PIIINP were all significantly increased in patients compared with controls. Neither myocardial nor plasma Gal-3 were correlated with myocardial collagen and circulating biomarkers; whereas PICP was correlated with myocardial total (r = 0.819, P < 0.001) and collagen type I (r = 0.744, P < 0.001) deposition, PIIINP was not. In the non-invasive study both plasma Gal-3 and serum PICP were increased (P < 0.001) in patients compared with controls. No correlation was found between Gal-3 and PICP in HF patients., Conclusions: These findings show that although an excess of cardiac and systemic Gal-3 is present in patients with HF of hypertensive origin, this molecule is not associated with histological, molecular and biochemical parameters related to myocardial fibrosis in these patients., (© 2015 The Authors. European Journal of Heart Failure © 2015 European Society of Cardiology.)

Cardiotrophin-1 has been shown to be profibrogenic in experimental models. The aim of this study was to analyze whether cardiotrophin-1 is associated with left ventricular end-diastolic stress and myocardial fibrosis in hypertensive patients with heart failure. Endomyocardial biopsies from patients (n=31) and necropsies from 7 control subjects were studied. Myocardial cardiotrophin-1 protein and mRNA and the fraction of myocardial volume occupied by collagen were increased in patients compared with controls (P<0.001). Cardiotrophin-1 overexpression in patients was localized in cardiomyocytes. Cardiotrophin-1 protein was correlated with collagen type I and III mRNAs (r=0.653, P<0.001; r=0.541, P<0.01) and proteins (r=0.588, P<0.001; r=0.556, P<0.005) in all subjects and with left ventricular end-diastolic wall stress (r=0.450; P<0.05) in patients. Plasma cardiotrophin-1 and N-terminal pro-brain natriuretic peptide and serum biomarkers of myocardial fibrosis (carboxy-terminal propeptide of procollagen type I and amino-terminal propeptide of procollagen type III) were increased (P<0.001) in patients compared with controls. Plasma cardiotrophin-1 was correlated with N-terminal pro-brain natriuretic peptide (r=0.386; P<0.005), carboxy-terminal propeptide of procollagen type I (r=0.550; P<0.001), and amino-terminal propeptide of procollagen type III (r=0.267; P<0.05) in all subjects. In vitro, cardiotrophin-1 stimulated the differentiation of human cardiac fibroblast to myofibroblasts (P<0.05) and the expression of procollagen type I (P<0.05) and III (P<0.01) mRNAs. These findings show that an excess of cardiotrophin-1 is associated with increased collagen in the myocardium of hypertensive patients with heart failure. It is proposed that exaggerated cardiomyocyte production of cardiotrophin-1 in response to increased left ventricular end-diastolic stress may contribute to fibrosis through stimulation of fibroblasts in heart failure of hypertensive origin.

We investigated whether the quality of myocardial collagen associates with elevated left-sided filling pressures in 38 hypertensive patients with stage C chronic heart failure. Filling pressures were assessed invasively measuring pulmonary capillary wedge pressure. Left ventricular chamber stiffness constant was calculated from the deceleration time of the early mitral filling wave. The fraction of myocardial volume occupied by total collagen tissue and collagen type I fibers was assessed histomorphologically. The degree of collagen cross-linking (CCL), which determines the formation of insoluble stiff collagen, was assessed by colorimetric and enzymatic procedures. The expression of lysyl oxidase (LOX), which regulates CCL, was assessed by Western blot. Compared with patients with normal pulmonary capillary wedge pressure (≤12 mm Hg; n=16), patients with elevated pulmonary capillary wedge pressure (>12 mm Hg; n=22) exhibited increases of left ventricular chamber stiffness constant, fraction of myocardial volume occupied by total collagen tissue, fraction of myocardial volume occupied by collagen type I fibers, CCL, insoluble stiff collagen, and LOX. Pulmonary capillary wedge pressure was correlated with left ventricular chamber stiffness constant (r=0.639; P<0.001), insoluble stiff collagen (r=0.474; P<0.005), CCL (r=0.625; P<0.001), and LOX (r=0.410; P<0.05) in all of the patients but not with fraction of myocardial volume occupied by total collagen tissue or fraction of myocardial volume occupied by collagen type I fibers. In addition, CCL was correlated with insoluble stiff collagen (r=0.612; P<0.005), LOX (r=0.538; P<0.01), left ventricular chamber stiffness constant (r=0.535; P<0.005), peak filling rate (r=-0.343; P<0.05), ejection fraction (r=-0.430; P<0.01), and amino-terminal propeptide of brain natriuretic peptide (r=0.421; P<0.05) in all of the patients. These associations were independent of confounding factors. These findings indicate that, in hypertensive patients with stage C heart failure, it is only the quality of collagen (ie, degree of cross-linking) that associates with elevated filling pressures. It is suggested that LOX-mediated excessive CCL facilitates the increase in left ventricular stiffness with the resulting elevation of filling pressures in these patients.

Aims: To investigate whether Annexin A5 (AnxA5) is related to hypertensive heart disease (HHD) and whether this relation is dependent of apoptosis., Methods and Results: Hypertensives without cardiac abnormalities (stage A), with left ventricular hypertrophy (LVH) (stage B), and with LVH and clinical manifestations of chronic HF (stage C), were studied. AnxA5 was quantified in endomyocardial biopsies by real time RT-PCR, Western blot, and immunohistochemistry, and apoptosis by DNA fragmentation, caspase-3 activation, and PARP and Bax/Bcl-2 ratios. Plasma AnxA5 was measured by ELISA in samples from the coronary sinus and the antecubital vein. Although AnxA5 mRNA did not change, myocardial and plasma AnxA5 were increased in hypertensives stages B and C compared with normotensives and hypertensives stage A. Myocardial AnxA5 was inversely correlated with parameters assessing systolic function in all hypertensives, this association being independent of apoptosis. Myocardial AnxA5 was directly correlated with plasma AnxA5. Plasma AnxA5 was inversely correlated with systolic function in all hypertensives., Conclusion: This cross-sectional study shows that myocardial AnxA5 upregulation is associated with HHD and impairment of systolic function in hypertensive patients, this association being independent of apoptosis. Plasma AnxA5 can be a marker of myocardial AnxA5 upregulation and systolic dysfunction in patients with HHD.

Objective: To investigate whether the glycoprotein (gp130)-mediated survival pathway, which protects cardiomyocytes from apoptosis, is depressed in left ventricular hypertrophy hypertensive patients with chronic heart failure., Methods: Transvenous endomyocardial biopsies were obtained in 52 hypertensive patients with left ventricular hypertrophy: 28 without heart failure and 24 with heart failure. gp130 and gp130-dependent antiapoptotic pathways p42/44 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) as well as gp130 agonist cardiotrophin-1 were analyzed by reverse transcriptase-polymerase chain reaction and western blot. Apoptosis was assessed by DNA end-labeling (TUNEL), caspase-3 immunostaining and caspase substrate poly(ADP-ribose) polymerase cleavage., Results: gp130 protein expression (P < 0.05) and p42/44 MAPK and PI3K/Akt activation (P < 0.01) were decreased in heart-failure hypertensive patients compared with nonheart-failure hypertensive individuals. No changes in gp130 mRNA expression were found between the two groups. Cardiotrophin-1 was increased (P < 0.05) at both the mRNA and protein levels in heart-failure hypertensive individuals compared with nonheart-failure hypertensive individuals. Cardiomyocyte apoptosis was increased (P < 0.01) in heart-failure hypertensive individuals compared with nonheart-failure hypertensive individuals. Inverse correlations (P < 0.01) occurred between cardiomyocyte apoptosis and p42/44 MAPK and PI3K/Akt activation in all hypertensive patients. Cardiotrophin-1 correlated inversely (r = -0.554, P < 0.05) with gp130 in all hypertensive individuals. In cultured HL-1 cardiomyocytes, cardiotrophin-1 decreased (P < 0.05) the gp130:phosphorylated gp130 (at Ser782) ratio and increased (P < 0.05) gp130ubiquitination., Conclusions: An association exists between depression of the gp130 cytoprotective pathway and stimulation of cardiomyocyte apoptosis in hypertensive patients that develop heart failure. Whether the excess of cardiotrophin-1 induces ligand-induced receptor down-regulation in these patients requires further study.

Objectives: We sought to assess the distribution of collagen deposits and collagen degradation in hypertensive patients with either systolic heart failure (SHF) or diastolic heart failure (DHF)., Background: Increased collagen synthesis and deposition have been described in the myocardium of heart failure (HF) hypertensive patients., Methods: We studied 39 HF hypertensive patients subdivided into two groups: 16 with SHF and 23 with DHF. Endomyocardial biopsies were performed to quantify mysial (i.e., perimysial plus endomysial) and perivascular and scar-related collagen volume fraction (CVF). Matrix metalloproteinase (MMP)-1 and its tissue inhibitor matrix metalloproteinase (TIMP)-1 were analyzed in cardiac samples by Western blot and immunohistochemistry, and in blood samples by enzyme-linked immunosorbent assay., Results: Mysial CVF was lower in SHF hypertensive patients than in normotensive (p < 0.05) and DHF hypertensive patients (p < 0.01). Perivascular and scar-related CVF was higher (p < 0.05) in the two groups of hypertensive patients than in normotensive subjects, and in SHF hypertensive compared with DHF hypertensive patients. The MMP-1:TIMP-1 ratio was increased (p < 0.05) in tissue and serum samples from the SHF hypertensive group compared with the other two groups of subjects. The MMP-1 expression was increased (p < 0.01) in the interstitium and cardiomyocytes of SHF hypertensive patients compared with DHF hypertensive and normotensive subjects. The serum MMP-1:TIMP-1 ratio was inversely correlated with ejection fraction (r = -0.510, p < 0.001) and directly correlated with left ventricular end-diastolic diameter (r = 0.549, p < 0.001) in all subjects., Conclusions: These findings show that the pattern of collagen deposits and the balance of the MMP-1/TIMP-1 system are different in the myocardium of SHF and DHF hypertensive patients. It is proposed that excessive degradation of mysial collagen may be related to the compromise of systolic function in HF hypertensive patients.

Objective: To investigate whether cardiac expression of the nuclear peroxisome proliferator-activated receptor alpha (PPARalpha) is altered in patients with hypertensive heart disease (HHD)., Methods: We studied endomyocardial septal biopsies from 24 patients with essential hypertension divided into three groups: 6 without left ventricular hypertrophy (LVH) (HT group), 10 with LVH (LVH group), and 8 with LVH and heart failure (HF) (HF group). The expression of two PPARalpha isoforms (the native active and the truncated inhibitory) was analyzed by Western blot and reverse transcription polymerase chain reaction (RT-PCR), and two PPARalpha target genes were evaluated by RT-PCR. Histomorphological features were evaluated in a second myocardial sample from LVH and HF groups., Results: Whereas the expression of native PPARalpha protein was lower (p<0.05) in LVH and HF groups than in the HT group, truncated PPARalpha protein was overexpressed (p<0.001) in the HF group as compared with LVH and HT groups. The mRNA expression of native and truncated PPARalpha was similar in the three groups of hypertensives. In addition, a progressive decrease (p for trend<0.05) in the two PPARalpha target genes mRNA expression was observed among HT, LVH and HF groups. The amount of truncated PPARalpha protein correlates directly with cardiomyocytes apoptosis and inversely with cardiomyocytes density in patients with HHD. In addition, the expression of truncated PPARalpha protein was directly correlated with left ventricular volumes, and inversely with ejection fraction in all hypertensives., Conclusions: These findings suggest that post-transcriptional regulation of PPARalpha isoforms is altered in patients with HHD, namely in those developing HF. An excess of the truncated inhibitory isoform may be involved in hypertensive left ventricular failure and remodeling.

Background: We investigated whether increased collagen type I synthesis and deposition contribute to enhancement of myocardial fibrosis and deterioration of cardiac function in patients with hypertensive heart disease (HHD)., Methods and Results: We studied 65 hypertensives with left ventricular hypertrophy subdivided into 2 groups: 34 patients without heart failure (HF) and 31 patients with HF. Transvenous endomyocardial biopsies of the interventricular septum were performed to quantify the amount of fibrotic tissue and the extent of collagen type I deposition. The carboxy-terminal propeptide of procollagen type I (PIP), an index of collagen type I synthesis, was measured by radioimmunoassay in serum samples from the coronary sinus and the antecubital vein. Compared with normotensives, the amount of collagen tissue, the extent of collagen type I deposition, and coronary and peripheral PIP were increased (P<0.01) in the 2 groups of hypertensives. These parameters were also increased (P<0.01) in HF hypertensives compared with non-HF hypertensives. Coronary PIP was higher (P<0.01) than peripheral PIP in hypertensives but not in normotensives. The amount of collagen tissue was inversely correlated with the ejection fraction and directly correlated with both coronary and peripheral PIP in all hypertensives., Conclusions: These findings suggest that an excess of cardiac collagen type I synthesis and deposition may be involved in the enhancement of myocardial fibrosis that accompanies the development of HF in HHD. In addition, our data show that the heart secretes PIP via the coronary sinus into the peripheral circulation in patients with HHD. Thus, PIP determined in peripheral blood can be a useful marker of myocardial fibrosis in these patients.

Background: This study was designed to investigate whether myocardial collagen content is related to myocardial stiffness in patients with essential hypertension., Methods and Results: The study was performed in 34 patients with hypertensive heart disease. Nineteen of these patients were also evaluated after 12 months of treatment with losartan. Transvenous endomyocardial biopsies of the interventricular septum were performed to quantify collagen volume fraction (CVF). Left ventricular (LV) chamber stiffness (K(LV)) was determined from the deceleration time of the early mitral filling wave as measured by Doppler echocardiography. Histological analysis at baseline revealed the presence of 2 subgroups of patients: 8 with severe fibrosis and 26 with nonsevere fibrosis. Values of CVF and K(LV) were significantly higher in the 2 subgroups of hypertensives than in normotensives. In addition, compared with patients with nonsevere fibrosis, patients with severe fibrosis exhibited significantly increased values of CVF and K(LV). After treatment, CVF and K(LV) decreased significantly in patients with severe fibrosis (n=7). None of these parameters changed significantly after treatment in patients with nonsevere fibrosis (n=12). CVF was directly correlated with K(LV) (r=0.415, P<0.02) in all hypertensives., Conclusions: These findings show a strong association between myocardial collagen content and LV chamber stiffness in patients with essential hypertension. Our results also suggest that the ability of losartan to induce regression of severe myocardial fibrosis is associated with diminution of myocardial stiffness in hypertensive patients.

We investigated whether cardiac apoptosis is stimulated in the heart of hypertensive patients and whether angiotensin II plays a role in such alteration. The study was performed in 28 patients with essential hypertension and no evidence of either ischemic cardiomyopathy or heart failure. After randomization, 14 patients were assigned to losartan and 14 patients to amlodipine treatment. At baseline and after 12 months, right septal endomyocardial biopsies were performed, and the number of apoptotic nuclei was assessed by DNA end-labeling (TUNEL). In addition, immunostaining for the active form of caspase-3 was also performed to assess apoptosis. Compared with normotensive autopsied hearts, both cardiomyocyte and noncardiomyocyte apoptosis were increased (P<0.001) in hypertensive hearts. Time-course changes in blood pressure during treatment were similar in the 2 groups of patients. In losartan-treated patients, both cardiomyocyte and noncardiomyocyte apoptosis decreased (P<0.05). Neither cardiomyocyte nor noncardiomyocyte apoptosis changed significantly in amlodipine-treated patients. These findings indicate that apoptosis is abnormally stimulated in the heart of patients with essential hypertension. Our data also suggest that the ability of antihypertensive treatment to inhibit cardiac apoptosis is independent of its antihypertensive efficacy. We propose that angiotensin II may participate in the stimulation of cardiac apoptosis in essential hypertension.

Background: Excessive myocardial collagen cross-linking (CCL) determines myocardial collagen's resistance to degradation by matrix metalloproteinase (MMP)-1 and interstitial accumulation of collagen fibers with impairment of cardiac function.Objectives: This study sought to investigate whether CCL and a newly identified biomarker of this alteration are associated with hospitalization for heart failure (HHF) or cardiovascular death in patients with HF and arterial hypertension in whom other comorbidities were excluded.Methods: Endomyocardial biopsies and blood samples from 38 patients (invasive study), and blood samples from 203 patients (noninvasive study) were analyzed. Mean follow-ups were 7.74 ± 0.58 years and 4.72 ± 0.11 years, respectively. Myocardial CCL was calculated as the ratio between insoluble and soluble collagen. The ratio between the C-terminal telopeptide of collagen type I (CITP) and matrix metalloproteinase-1 (CITP:MMP-1) was determined in blood samples.Results: Invasive study: CCL was increased (p < 0.001) in patients compared with controls. Patients were categorized according to normal or high CCL values. Patients with high CCL exhibited higher risk for subsequent HHF (log-rank test p = 0.022), but not for cardiovascular death. CITP:MMP-1 was inversely associated with CCL (r = -0.460; p = 0.005) in all patients. Receiver operating characteristic curves rendered a CITP:MMP-1 cutoff ≤1.968 (80% sensitivity and 76% specificity) for predicting high CCL. Noninvasive study: Patients were categorized according to CITP:MMP-1 ratio values as normal ratio (>1.968) or low ratio (≤1.968). Patients with a low ratio exhibited higher risk for HHF (log-rank test p = 0.014), which remained significant after adjustment for relevant covariables (adjusted hazard ratio: 2.22; 95% CI: 1.37 to 3.59, p = 0.001). In addition, CITP:MMP-1-based categorization yielded significant integrated discrimination and net reclassification improvements (p = 0.003 and p = 0.009, respectively) for HHF over relevant risk factors. CITP:MMP-1 was not associated with the risk of cardiovascular death.Conclusions: Excessive myocardial CCL is associated with HHF in hypertensive patients with HF. In this population, the serum CITP:MMP-1 ratio identifies patients with increased CCL and high risk of HHF. [ABSTRACT FROM AUTHOR]