Oleanolic acid: a novel cardioprotective agent that blunts hyperglycemia-induced contractile dysfunction

Diabetes constitutes a major health challenge. Since cardiovascular complications are common in diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher in-hospital mortality. Previous studies implicate oxidative stress, excessive flux through the hexosamine biosynthetic pathway (HBP) and a dysfunctional ubiquitin-proteasome system (UPS) as potential mediators of this process. Since oleanolic acid (OA; a clove extract) possesses antioxidant properties, we hypothesized that it attenuates acute and chronic hyperglycemia-mediated pathophysiologic molecular events (oxidative stress, apoptosis, HBP, UPS) and thereby improves contractile function in response to ischemia-reperfusion. We employed several experimental systems: 1) H9c2 cardiac myoblasts were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with two OA doses (20 and 50 µM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 min reperfusion ± OA treatment; 3) In vivo coronary ligations were performed on streptozotocin treated rats ± OA administration during reperfusion; and 4) Effects of long-term OA treatment (2 weeks) on heart function was assessed in streptozotocin-treated rats. Our data demonstrate that OA treatment blunted high glucose-induced oxidative stress and apoptosis in heart cells. OA therapy also resulted in cardioprotection, i.e. for ex vivo and in vivo rat hearts exposed to ischemia-reperfusion under hyperglycemic conditions. In parallel, we found decreased oxidative stress, apoptosis, HBP flux and proteasomal activity following ischemia-reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. These findings are promising since it may eventually result in novel therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients) and diabetic patients with associated cardiovascular complications

Natural reversal of pulmonary vascular remodeling and right ventricular remodeling in SU5416/hypoxia-treated Sprague-Dawley rats.

Pulmonary arterial hypertension (PAH) is a lethal disease and improved therapeutic strategies are needed. Increased pulmonary arterial pressure, due to vasoconstriction and vascular remodeling, causes right ventricle (RV) failure and death in patients. The treatment of Sprague-Dawley rats with SU5416 injection and exposure to chronic hypoxia for three weeks followed by maintenance in normoxia promote progressive and severe PAH with pathologic features that resemble human PAH. At 5-17 weeks after the SU5416 injection, PAH is developed with pulmonary vascular remodeling as well as RV hypertrophy and fibrosis. The present study investigated subsequent events that occur in these PAH animals.At 35 weeks after the SU5416 injection, rats still maintained high RV pressure, but pulmonary vascular remodeling was significantly reduced. Metabolomics analysis revealed that lungs of normal rats and rats from the 35-week time point had different metabolomics profiles. Despite the maintenance of high RV pressure, fibrosis was resolved at 35-weeks. Masson's trichrome stain and Western blotting monitoring collagen 1 determined 12% fibrosis in the RV at 17-weeks, and this was decreased to 5% at 35-weeks. The level of myofibroblasts was elevated at 17-weeks and normalized at 35-weeks.These results suggest that biological systems possess natural ways to resolve pulmonary and RV remodeling. The resolution of RV fibrosis appears to involve the reduction of myofibroblast-dependent collagen synthesis. Understanding these endogenous mechanisms should help improve therapeutic strategies to treat PAH and RV failure

Metabolomics analysis of the lung.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 5 weeks (8-wks time point) or 32 weeks (35-wks time point). Metabolomics analysis was performed in the lung tissues. Volcano plots (on the left) and Partial least squares Discriminant Analysis (PLS-DA; on the right) for (A) 8-weeks PAH (green circles) vs. normal control (red circles), (B) 8-weeks PAH (green circles) vs. 35-weeks PAH (red circles) and (C) 35-weeks PAH (green circles) vs. normal control (red circles) are shown. Dot volcano plots represent fold changes (FC) on the x-axis and the statistical significance in <i>P</i> value on the y-axis. Pink dots represent values with <i>P</i>≤0.05 and changes of greater than twofold.</p

RV hemodynamics.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 2, 5, 14 or 32 weeks (5-, 8-, 17- and 35-wks time points, respectively). Animals were anaesthetized, ventilated and the chest opened. A Millar catheter was inserted into the RV apex and hemodynamic measurements were performed. (A) Flow diagram of the experiments. (B) Representative hemodynamic traces. (C) Means ± SEM of RVSP. (D) Means ± SEM of dP/dt_max. (E) Means ± SEM of the ratio of RV weight to LV + septum weight. *Significantly different from each other at <i>P</i> < 0.05. **Significantly different from each other at <i>P</i> < 0.01. ***Significantly different from each other at <i>P</i> < 0.001.</p

RV damage and fibrosis.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 2, 5, 14 or 32 weeks (5-, 8-, 17- and 35-wks time points, respectively). (A) Representative H&E staining results of the RV tissue sections. (B) Masson’s trichrome stain results in the RVs. Blue stains indicate fibrotic areas. Bar graph represents means ± SEM of % fibrotic area (N = 7). *Significantly different from each other at <i>P</i> < 0.05. **Significantly different from each other at <i>P</i> < 0.01. (C) Western blotting showing COLA1 expression as an indicator of fibrosis in the RVs at 17 and 35 weeks after the SU5416 injection. Bar graph represents means ± SEM of the ratio of COLA1 to G3PDH. *Significantly different from the control value.</p

Lung structure.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 14 or 32 weeks (17- and 35-wks time points, respectively). The large panels on the left are representative VVG stains at x100 magnification. Sections of different sizes of pulmonary arteries (50-150nm) are enlarged (x1000) on the right, showing VVG stain, Masson’s trichrome (MT) stain and immunohistochemistry of α-smooth muscle actin (αSMA).</p

Myofibroblasts in the RV.

<p>SU5416-injected rats were subjected to 3 weeks in hypoxia and then maintained in normoxia for 14 or 32 weeks (17- and 35-wks time points, respectively). Hearts were fixed in formalin and embedded in paraffin. Longitudinal sections of the RV myocardium were subjected to immunohistochemistry using the antibody against (A) α-smooth muscle actin and (B) periostin. Results are presented at x200 and x1,000 magnifications. Scale bars indicate 200 μm for x200 and 50 μm for x1,000. The brown stains indicate the expression of α-smooth muscle actin or periostin. Arrows depict that neither α-smooth muscle actin nor periostin stains are present in fibroblasts of the normal RV; brown α-smooth muscle actin and periostin stains are present overlapping nuclear stains of fibroblasts at 17-wks; and α-smooth muscle actin and periostin stains in fibroblasts are absent at 35-wks.</p

OA treatment decreases infarct size following coronary artery ligation in streptozotocin-diabetic rats.

<p>Wistar rats were injected with STZ and followed for a 1-week period. Subsequently, 0.45 mg/kg OA was injected via the penile vein within the first two min of reperfusion. Evans blue dye and TTC staining enabled visualization of viable tissue (blue), infarcted area (white) and the area at risk (red). Values are expressed as mean ± SEM (n = 6). <b>*</b>p<0.05, **p<0.01 vs. respective controls.</p

OA treatment attenuates <i>O</i>-GlcNAcylation in hearts subjected to ischemia-reperfusion under high glucose conditions.

<p>Isolated rat hearts were perfused under high glucose conditions vs. controls and subjected to ischemia-reperfusion ± OA treatment during reperfusion. Western blot analysis for overall <i>O</i>-GlcNAcylation is shown with β–actin as loading control. Densitometric analysis for <i>O</i>-GlcNAcylation is displayed below gel image (normalized to corresponding β–actin values). Values are expressed as mean ± SEM (n = 6). <b>*</b>p<0.05, **p<0.01 vs. respective controls.</p