Role of Rho-Kinase in the Pathogenesis of Hypertension: Therapeutic Potential of Rho- Kinase Inhibitors in Hypertension

Yoshitaka Hirookaa
Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

The Rho-kinase pathway appears to be involved in the pathogenesis of hypertension, and Rho-kinase inhibitors such as fasudil may have potential as antihypertensive agents, according to studies from this group of investigators.

The Rho-kinase pathway is involved in various functions, including calcium sensitization and contraction of vascular smooth muscle cells. Several key findings from these investigators point to a role for Rho-kinase in hypertension. First, in the spontaneously hypertensive rat model, upregulation of Rho-kinase in blood vessels was associated with vascular hyper-reactivity and this preceded the development of hypertension. In spontaneously hypertensive rats (SHR) as well as angiotensin II-induced hypertensive rats, Rho-kinase was substantially involved in vascular lesion formation, and the Rho-specific inhibitor fasudil inhibited vascular lesion formation.

Secondly, activation of the Rho-kinase pathway occurs within the central nervous system and is associated with hypertension. SHR showed increased membrane expression of Rho-A and activity of Rho-kinase in the brainstem, and blockade of Rho-kinase expression with Rho-kinase inhibitors (Y-27632 or fasudil) produced a decrease in blood pressure and sympathetic nerve activity. Normal Wistar-Kyoto rats demonstrated less expression and less effect on blood pressure with Rho-kinase inhibitors. Similar findings were also demonstrated in an experiment using adenovirus vector encoding dominant-negative Rho-kinase (Figures 1 and 2).

Finally, in controlled studies in humans, fasudil increased forearm blood flow in patients with essential hypertension, but not in normotensive subjects, and it normalized angiotensin-II-induced forearm vasoconstriction in normotensive subjects.

Altogether, these observations indicate that Rho-kinase is substantially involved in the mechanism of hypertension related to the activation of the renin-angiotensin system and the sympathetic nervous system. These data suggest there may be a therapeutic role for Rho-kinase inhibitors, which appear to preferentially mediate the hypertensive state.

Morning Blood Pressure Surge and Associated Risk Factors for Cardiovascular Disease in Hypertension

Kazuomi Kario
Jichi Medical School, Tochigi, Japan

A morning surge in blood pressure is associated with several cardiovascular disease-related risk factors and an increased likelihood of stroke. Previous studies have shown that the peak incidence of cardiovascular events is in the morning hours, a time at which there are also increases in ambulatory blood pressure (BP) levels and tissue-type plasminogen activator inhibitor-1 (PAI-1). These investigators evaluated the relationship between these factors and cardiovascular risk in 519 hypertensive patients (mean age 72) without cardiovascular disease at baseline. Patients underwent extensive ambulatory BP studies, including assessment of morning surge blood pressure (MS) and brain MRI to identify silent cerebral infarcts.  
 
Study subjects were classified as having MS (cut-off of 55 mm Hg) or no MS. By MRI analysis, the MS group had a higher prevalence and greater number of silent cerebral infarcts at baseline. At a mean follow-up of 41 months, stroke incidence was also higher in the MS group, with a relative risk of 2.7 independent of age and 24-hour systolic BP level. Morning onset of stroke was almost twice as common in the MS group as the non-MS group. Thus, in hypertensives, exaggerated MS was associated with an independent risk for hypertensive target organ damage and cardiovascular events, Kario reported.
 
As a possible source for the MS, the rate of orthostatic hypertension was greatly increased among MS subjects, as was systolic and diastolic BP, in a study sample. Subjects with orthostatic hypertension or hypotension, compared with normotensives, also had a greater prevalence of multiple lacuna infarcts. Exaggerated MS, therefore, was associated with orthostatic hypertension, sympathetic hyperactivity, and increased ambulatory BP variability.
 
Tests of platelet aggregation also showed significantly higher spontaneous small-platelet-aggregates in the morning in MS subjects, versus those without MS (103 vs 42 x 103 counts; p <0.0001), but no significant differences in the evening. PAI-1 levels were not higher in MS subjects than in non-MS subjects. Patients with multiple silent infarcts also had higher levels of small-size platelet aggregates, PAI-1, and indicators of thrombin generation and endothelial stimulation.
 
“The exaggerated morning blood pressure surge and morning hypertension could be a new therapeutic target for more effective prevention of cardiovascular events in hypertensive patients,” Kario stated.

Candesartan Can Reduce Cardiovascular Events By Decreasing Sympathetic Nerve Activity and Plasma Aldosterone

Hiroo Kumagai
Keio University School of Medicine, Tokyo, Japan

The angiotensin receptor blocker (ARB) candesartan was shown to affect a number of factors that relate to increased cardiovascular risk and target organ damage from hypertension, in a series of studies presented by Hiroo Kumagai and colleagues from Keio University School of Medicine, Tokyo.
 
When neurons of the rostral ventrolateral medulla oblongata (RVLM), the sympathetic center, of spontaneously hypertensive rats were superfused with candesartan, the ARB hyperpolarized the membrane potential of the neurons and reduced the firing rates, outcomes that did not occur in normotensive rats. Infusions of candesartan also prevented an increase in renal sympathetic nerve activity (SNA) when blood pressure was experimentally decreased in SHR rats. In an additional experiment, 2 weeks administration of candesartan produced significant suppression of renal SNA in the rats, versus vehicle, despite significant increases in blood pressure and renal blood flow. This experiment, along with previous findings in humans, “strongly indicates that candesartan can reduce peripheral SNA,” Kumagai said.
 
Keio University researchers also found that candesartan improved disorders of cardiovascular regulation. Normal neurocardiovascular regulation (heart rate) is characterized by low linearity and high non-linearity (ie, high complexity) and is chaotic and dynamic, while abnormal regulation of the cardiovascular system is characterized by high linearity and low non-linearity (low complexity). Patients with a reduced non-linear component of the heart rate are at increased risk for cardiovascular events, he said, as background.
 
In the current study, the cardiovascular system of SHR was characterized by high linearity and low non-linearity, and candesartan improved the linearity and therefore the cardiovascular regulation.

Finally, the effect of candesartan on aldosterone levels was evaluated in a 2-year prospective study of 46 patients with glomerulonephritis. Both the ACE inhibitor and candesartan reduced blood pressure similarly, but the ARB produced greater reductions in proteinuria, plasma aldosterone concentrations (Figure 1), and SNA. The reduction in aldosterone and potentiated SNA should be protective against renal interstitial fibrosis and other target organ damage, therefore, candesartan may have renovascular and cardiovascular protective actions, Dr. Kumagai said. Figure 2 shows the mechanisms by which ARBs are renoprotective.

Hypertension and Endothelial Function: Role of Angiotensin II and Mechanical Pressure in Oxidative Stress

Yukihito Higashi
Graduate School of Biomedical Sciences Hiroshima University, Hiroshima, Japan

Investigators from Hiroshima University examined the mechanisms by which hypertension alters endothelial function, and demonstrated the role of oxidative stress induced by the activation of the renin-angiotensin system.
 
As background, they noted recent reports that angiotensin II stimulates the production of reactive oxygen species through the activation of membrane-bound NADH/NADPH oxidase (Figure 1). Endothelial dysfunction may be promoted when an imbalance occurs: reduced production of nitric oxide (NO) or increased production of reactive oxygen species, mainly superoxide.

Patients with renovascular hypertension serve as good models for determining how endothelium-dependent vasodilation is affected by excess angiotensin II and angiotensin II-related increases in oxidative stress. Such patients have impaired endothelium-dependent vasodilation induced by acetylcholine. In the study presented by Higashi, this impairment (enhanced response to acetylcholine) was restored by renal angioplasty and by the infusion of the antioxidant vitamin C. Furthermore, the improvement of endothelial function correlated with the reduction in markers of oxidative stress, such as 8-OHdG and MDA-LDL.

In addition, ACE inhibitors and angiotensin II receptor blockers (ARBs) improved endothelial dysfunction of forearm and renal circulation in patients with hypertension. The effect was similar between the ACE inhibitor and ARBs. Other antihypertensives (calcium antagonists, beta blockers and diuretics) had no significant effect on endothelial dysfunction, as judged by blood flow. The improvement in endothelial function after angioplasty or medication, however, did not correlate with a reduction in blood pressure, the study also found.
 
These findings suggest that excess oxidative stress generated by angiotensin II is involved, at least in part, in impaired endothelium-dependent vasodilation and that change in mechanical pressure generated by high blood pressure may not directly promote the restoration of endothelial function in human hypertension. ACE inhibitors and ARBs augment endothelial function in hypertensive patients, possibly due to NO production. These agents should be able to improve endothelial dysfunction and thus prevent cardiovascular complications, Dr. Higashi said.

Mechanism of Transition from Hypertrophy to Heart Failure

Tohru Masuyama
Osaka University Graduate School of Medicine, Suita, Japan

Long-standing hypertension leads to left ventricular hypertrophy (LVH), which leads to diastolic dysfunction and progresses to diastolic heart failure (DHF). The paucity of data on DHF is largely due to the lack of an animal model, but Tohru Masuyama and colleagues at Osaka University Graduate School have developed a model in which compensatory LVH develops early and progresses to heart failure (HF) by 90 weeks of age. In this model, left ventricular diastolic dysfunction was related to structural changes, progressive myocardial hypertrophy, and ultimately myocardial fibrosis.
 
“In our model, hypertension caused structural abnormalities, such as LVH and myocardial fibrosis. The development of these structural abnormalities is considered to lead to diastolic failure and thus to DHF,” Dr. Masuyama said. Interestingly, treatment with an angiotensin receptor blocker (ARB) but not a calcineurin inhibitor prevented myocardial fibrosis.
 
Additionally, blood pressure patterns in the DHF model were normal in the compensatory LVH stage but altered in the HF stage. Increases in left ventricular end diastolic pressure were observed only in the advanced stage. The effects of blood pressure lowering on myocardial fibrosis were minimal.
 
While LVH has been considered an adaptive response to pressure overload, experiments in this model did not demonstrate a direct effect. The structural changes were closely related, however, to neurohumoral activation; thus, neurohumoral activation appears to aid in the transition from compensatory LVH to HF, he said.
 
The maladaptive LVH appears to be induced independently of mechanical stress and by the activation of growth factors and agonists binding to G-protein-coupled receptors. Induction of these is mediated by the shedding of heparin-binding epidermal growth factor (HB-EGF) and/or by the generation of reactive oxygen species. Alteration in Ca2+ handling in the hypertrophied myocyte is also likely involved in the development of LVH itself as well as in the myocyte relaxation abnormality. The new inhibitor of HB-EGF shedding, KBR 7785, blocks the shedding of HB-EGF and attenuates cardiac hypertrophy, suggesting a therapeutic role for this agent.
 
In addition, maladaptive LVH in the model was associated with enhanced interstitial fibrosis and activation of matrix metalloproteinases (MMPs). MMP activity was increased in DHF and was normalized by administration of ARBs. Long-term administration of ARBs also inactivated collagen type 1 and type 3 production, which may translate into a reduction in fibrosis with ARB therapy, he said.

In systolic heart failure (SHF), gelatin zymography demonstrated enhancement of MMP2 activity before left ventricular dilatation, which was suppressed by chronic ACE inhibition, but no enhancement of MMP9 activity (Figure 1). After LV dilatation, both MMP2 and MMP9 were activated; ACE inhibition suppressed both MMP2 and MMP9 and prevented LV remodeling. “Thus, MMP9 rather than MMP2 may be involved in LV dilatation in SHF and could be a target for preventing LV remodeling,” he said.