Klaudia Budzyn

Therapeutic Targeting of Mitochondrial Superoxide in Hypertension

Rationale: Superoxide (&OV0151;) has been implicated in the pathogenesis of many human diseases including hypertension; however, commonly used antioxidants have proven ineffective in clinical trials. It is possible that these agents are not adequately delivered to the subcellular sites of superoxide production. Objective: Because the mitochondria are important sources of reactive oxygen species, we postulated that mitochondrial targeting of superoxide scavenging would have therapeutic benefit. Methods and Results: In this study, we found that the hormone angiotensin (Ang II) increased endothelial mitochondrial superoxide production. Treatment with the mitochondria-targeted antioxidant mitoTEMPO decreased mitochondrial &OV0151;, inhibited the total cellular &OV0151;, reduced cellular NADPH oxidase activity, and restored the level of bioavailable NO. These effects were mimicked by overexpressing the mitochondrial MnSOD (SOD2), whereas SOD2 depletion with small interfering RNA increased both basal and Ang II–stimulated cellular &OV0151;. Treatment of mice in vivo with mitoTEMPO attenuated hypertension when given at the onset of Ang II infusion and decreased blood pressure by 30 mm Hg following establishment of both Ang II–induced and DOCA salt hypertension, whereas a similar dose of nontargeted TEMPOL was not effective. In vivo, mitoTEMPO decreased vascular &OV0151;, increased vascular NO production and improved endothelial-dependent relaxation. Interestingly, transgenic mice overexpressing mitochondrial SOD2 demonstrated attenuated Ang II–induced hypertension and vascular oxidative stress similar to mice treated with mitoTEMPO. Conclusions: These studies show that mitochondrial &OV0151; is important for the development of hypertension and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in this and possibly other diseases.

Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.

Atherosclerosis is closely associated with disturbed flow characterized by low and oscillatory shear stress, but studies directly linking disturbed flow to atherogenesis is lacking. The major reason for this has been a lack of an animal model in which disturbed flow can be acutely induced and cause atherosclerosis. Here, we characterize partial carotid ligation as a model of disturbed flow with characteristics of low and oscillatory wall shear stress. We also describe a method of isolating intimal RNA in sufficient quantity from mouse carotid arteries. Using this model and method, we found that partial ligation causes upregulation of proatherogenic genes, downregulation of antiatherogenic genes, endothelial dysfunction, and rapid atherosclerosis in 2 wk in a p47(phox)-dependent manner and advanced lesions by 4 wk. We found that partial ligation results in endothelial dysfunction, rapid atherosclerosis, and advanced lesion development in a physiologically relevant model of disturbed flow. It also allows for easy and rapid intimal RNA isolation. This novel model and method could be used for genome-wide studies to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases.

Reversal of Vascular Macrophage Accumulation and Hypertension by a CCR2 Antagonist in Deoxycorticosterone/Salt-Treated Mice

Infiltration of macrophages into the artery wall plays detrimental roles during hypertension by promoting vascular inflammation and endothelial dysfunction, and it occurs via a chemo-attractant action of chemokines on macrophage cytokine receptors. We sought to identify the key chemokine receptors associated with macrophage infiltration into the vascular wall during deoxycorticosterone acetate (DOCA)/salt-induced hypertension in mice and to evaluate the impact of pharmacological inhibition of these receptors on blood pressure and leukocyte accumulation. Mice treated with DOCA/salt for 21 days displayed markedly elevated systolic blood pressure (158±2 versus 114±5 mm Hg in sham group; P<0.0001). Polymerase chain reaction screening via a gene array of 20 chemokine receptors indicated an increased expression of CCR2 in aortas of DOCA/salt-treated mice. Real-time polymerase chain reaction confirmed mRNA upregulation of CCR2 in aortas from DOCA/salt-treated animals and of the CCR2 ligands CCL2, CCL7, CCL8, and CCL12 (all >2-fold versus sham; P<0.05). Flow cytometry revealed 2.9-fold higher macrophage numbers (ie, CD45+ CD11b+ F4/80+ cells) in the aortic wall of DOCA/salt versus sham-treated mice. Intervention with a CCR2 antagonist, INCB3344 (30 mg/kg per day, IP), 10 days after the induction of hypertension with DOCA/salt treatment, reduced the aortic expression of CCR2 mRNA and completely reversed the DOCA/salt-induced influx of macrophages. Importantly, INCB3344 substantially reduced the elevated blood pressure in DOCA/salt-treated mice. Hence, our findings highlight CCR2 as a promising therapeutic target to reduce both macrophage accumulation in the vascular wall and blood pressure in hypertension.

Evidence That Estrogen Suppresses Rho-Kinase Function in the Cerebral Circulation In Vivo

Background and Purpose— Premenopausal women are less susceptible to cardiovascular diseases than men or postmenopausal women. Such disease states are often associated with increased vascular RhoA/Rho-kinase activity and decreased activity of nitric oxide (NO). This study tested whether female gender is associated with lower Rho-kinase activity or higher NO activity in cerebral arteries in vivo and whether estrogen contributes to any such gender differences. Methods— Changes in basilar artery diameter were measured with the use of a cranial window preparation in anesthetized Sprague-Dawley rats. Some female rats were ovariectomized (OVX) and treated subcutaneously daily for 14 days with vehicle (dimethyl sulfoxide) or 17β-estradiol. Vascular expression of RhoA or Rho-kinase was assessed by Western blotting. Results— The Rho-kinase inhibitor Y-27632 was selectively ≈3-fold more potent as a cerebral vasodilator in males versus females. Expression of total RhoA or Rho-kinase did not differ between males and females. In OVX rats, vasodilator responses to Y-27632 resembled responses in males. Treatment of OVX rats with 17β-estradiol normalized the vasodilator effects of Y-27632 to be equivalent to responses in intact female controls. The NO synthase inhibitor N-nitro-l-arginine methyl ester caused ≈50% greater constriction of the basilar artery in females versus males, but responses in OVX rats treated with either vehicle or 17β-estradiol did not differ from those recorded in intact females. Conclusions— These data indicate that vascular Rho-kinase function is suppressed in females because of the effects of estrogen, whereas the higher NO activity in females is estrogen independent.

Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension

Recent studies have emphasized the role of perivascular inflammation in cardiovascular disease. We studied mechanisms of perivascular leukocyte infiltration in angiotensin II (Ang II)‐induced hypertension and their links to vascular dysfunction. Chronic Ang II infusion in mice increased immune cell content of T cells (255 ± 130 to 1664 ± 349 cells/mg; P < 0.01), M1 and M2 macrophages, and dendritic cells in perivascular adipose tissue. In particular, the content of T lymphocytes bearing CC chemokine receptor (CCR) 1, CCR3, and CCR5 receptors for RANTES chemokine was increased by Ang II (CCR1, 15.6 ± 1.5% vs. 31 ± 5%; P < 0.01). Hypertension was associated with an increase in perivascular adipose tissue expression of the chemokine RANTES (relative quantification, 1.2 ± 0.2 vs. 3.5 ± 1.1; P< 0.05), which induced T‐cell chemotaxis and vascular accumulation of T cells expressing the chemokine receptors CCR1, CCR3, and CCR5. Mechanistically, RANTES–/– knockout protected against vascular leukocyte, and in particular T lymphocyte infiltration (26 ± 5% in wild type Ang II vs. 15 ± 4% in RANTES–/–), which was associated with protection from endothelial dysfunction induced by Ang II. This effect was linked with diminished infiltration of IFN‐γ‐producing CD8+ and double‐negative CD3+CD4–CD8– T cells in perivascular space and reduced vascular oxidative stress while FoxP3+ T‐regulatory cells were unaltered. IFN‐γ ex vivo caused significant endothelial dysfunction, which was reduced by superoxide anion scavenging. In a human cohort, a significant inverse correlation was observed between circulating RANTES levels as a biomarker and vascular function measured as flow‐mediated dilatation (R = –0.3, P < 0.01) or endothelial injury marker von Willebrand factor (R= +0.3; P< 0.01). Thus, chemokine RANTES is important in the regulation of vascular dysfunction through modulation of perivascular inflammation.—Mikolajczyk, T. P., Nosalski, R., Szczepaniak, P., Budzyn, K., Osmenda, G., Skiba, D., Sagan, A., Wu, J., Vinh, A., Marvar, P. J., Guzik, B., Podolec, J., Drummond, G., Lob, H. E., Harrison, D. G., Guzik, T. J. Role of chemokine RANTES in the regulation of perivascular inflammation, T‐cell accumulation, and vascular dysfunction in hypertension. FASEB J. 30, 1987–1999 (2016). www.fasebj.org

Segmental Differences in the Roles of Rho-Kinase and Protein Kinase C in Mediating Vasoconstriction

Rho-kinase and protein kinase C (PKC) have each been reported to mediate vasoconstriction via calcium sensitization. However, the relative contributions of these two kinases to vascular contraction, and whether their roles vary between large and small arteries, are largely unknown. We therefore assessed the relative roles of rho-kinase and PKC in mediating vasoconstriction in arteries from three segments of the aortic and mesenteric vasculature. We studied contractile responses of rat isolated thoracic aorta (diameter ≈2 mm), superior mesenteric artery (SMA; ≈1.5 mm), and second order branches of the superior mesenteric artery (BMA; ≈300 μm). The roles of rho-kinase and PKC in mediating contractile responses to phenylephrine, 9,11-dideoxy-9,11-methanoepoxy prostaglandin F2α (U46619), and KCl were assessed by using the rho-kinase inhibitor R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cycloheaxanecarboxamide (Y-27632) (1 and 10 μM) and the PKC inhibitor 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (Ro 31-8220) (5 μM). Contractile responses of aorta and SMA were reduced by either 1 or 10 μM Y-27632 (P < 0.05), whereas responses of BMA were reduced by 10 μM (P < 0.05) but not 1 μM Y-27632. In contrast, Ro 31-8220 partly reduced contractile responses in aorta and SMA (P < 0.05), but it abolished responses of BMA (P < 0.05). Cotreatment with Y-27632 and Ro 31-8220 markedly attenuated contractile responses to phenylephrine and KCl in all vessels, but it had only a moderate inhibitory effect on responses to U46619 in aorta and SMA. Thus, contractile responses of the larger arteries can involve both rho-kinase and PKC to varying degrees. Conversely, contractile responses of small mesenteric resistance arteries seem to be mediated exclusively by PKC, with no apparent role for rho-kinase.

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

Mechanisms of augmented vasoconstriction induced by 5-hydroxytryptamine in aortic rings from spontaneously hypertensive rats

To test whether development of enhanced vasoconstriction to 5‐hydroxytryptamine (5‐HT; serotonin) in SHR was temporally related to hypertension, elevated vascular superoxide (O2−) levels, decreased NO bioavailability, or increased contractile effects of cyclooxygenase or rho‐kinase and/or PKC.

Reduced cerebrovascular remodeling and functional impairment in spontaneously hypertensive rats following combined treatment with suboptimal doses of telmisartan and ramipril: is less really more?

Systemic arterial hypertension, which is the major risk factor for stroke [1], is accompanied by structural remodeling of cerebral arteries that initially serves to increase vascular resistance, thereby protecting the brain microvasculature from elevated mean arterial pressure and reducing the immediate threat of hemorrhagic stroke [2]. However, with structural remodeling of the cerebral vasculature comes reduced vasodilator capacity [2]. This not only increases the likelihood of cerebral ischemia and thus the incidence of ischemic strokes in hypertensive individuals, but also compromises the ability of cerebral vessels to support reperfusion after stroke, thereby worsening outcomes [3].

17 CHEMOKINE RECEPTORS AS NOVEL PHARMACOLOGICAL TARGETS TO REDUCE BLOOD PRESSURE DURING EXPERIMENTAL HYPERTENSION IN MICE

Introduction & Aims: Leukocyte infiltration into the artery wall plays a pathophysiological role in hypertension by promoting vascular inflammation and endothelial dysfunction [1]. Chemokines are chemoattractant cytokines that bind to receptors on leukocytes and promote their migration into tissues. The aims of this study were to identify chemokine receptors that are upregulated in the vascular wall during deoxycorticosterone acetate (DOCA)/salt-induced hypertension in mice, and to evaluate the impact of pharmacological inhibition of these receptors on blood pressure (BP) and leukocyte accumulation. Methods & Results: Mice treated with DOCA/salt for 21 d displayed markedly elevated systolic BP (158 ± 2 mmHg vs 114 ± 5 mmHg sham-treatment; P<0.0001; n≥11). In DOCA/salt-treated mice, flow cytometry revealed a 2.4-fold increase in numbers of CD45+ leukocytes in the aortic wall (P<0.05; n≥5). PCR screening via a gene panel containing 20 different chemokine receptors suggested an increase in expression of CCR2 in aortas of DOCA/salt-treated mice (n = 3). Real-time PCR confirmed upregulation of CCR2 in aortas from DOCA/salt-treated animals, and also levels of the CCR2 ligands CCL2, CCL7, CCL8 and CCL12 (>2-fold for all genes; n≥7; P<0.05). Administration of a CCR2 antagonist, INCB3344 (30 mg/kg/d, i.p.), to DOCA/salt-treated mice, reduced elevated aortic expression of CCR2 by 55% (n = 8, P<0.05). INCB3344 also appeared to reduce leukocyte accumulation in the aortic wall by ∼50%, although this effect was not significant (P>0.05, n = 5). Importantly, INCB3344 reversed DOCA/salt-induced elevations in systolic BP by ∼45% (n = 11, P<0.01). Conclusions: Our findings highlight CCR2 as a promising therapeutic target to reduce leukocyte accumulation and BP in hypertension. ReferencesGuzik TJ, Hoch NE, Brown KA, McCann LA, Rahman A, Dikalov S, Goronzy J, Weyand C, Harrison DG. Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction. J Exp Med. 2007 Oct 1;204(10):2449-60.