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(Hypertension. 1997;30:1397-1402.)
© 1997 American Heart Association, Inc.

Articles

Monocyte Chemoattractant Protein-1 Expression in Aortic Tissues of Hypertensive Rats

Quinn Capers, IV; R. Wayne Alexander; Pingping Lou; Hector De Leon; Josiah N. Wilcox; Nobukazu Ishizaka; Adam B. Howard; ; W. Robert Taylor

From the Department of Medicine, Divisions of Cardiology (Q.C., R.W.A., P.L., N.I., A.B.H., W.R.T.) and Hematology (H.deL., J.N.W.), Emory University School of Medicine, and Atlanta Veterans Administration Hospital (P.L., W.R.T.), Atlanta, Ga.

Correspondence to W. Robert Taylor, MD, PhD, Emory University School of Medicine, Division of Cardiology, Woodruff Memorial Research Building, Suite 319, 1639 Pierce Dr, Atlanta, Ga 30322.

	Abstract

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Abstract Monocyte chemoattractant protein-1 (MCP-1), a potent monocyte chemoattractant synthesized by vascular cells and monocytes, has been proposed to be an important mediator of inflammatory responses in the arterial vasculature. It was recently demonstrated that hypertension is associated with an inflammatory response in the arterial wall. To determine the effect of hypertension on arterial MCP-1 expression, we induced hypertension in Sprague-Dawley rats by infusing angiotensin II (0.75 mg · kg^-1 · d^-1 SC) for 7 days. Using Northern blot analysis, we detected a 3.6-fold increase in MCP-1 mRNA in the aortas of hypertensive rats. When we normalized blood pressure in angiotensin II–treated rats through oral administration of the nonspecific vasodilator hydralazine (15 mg · kg^-1 · d^-1), aortic MCP-1 mRNA expression was significantly reduced. Similar results were obtained with a norepinephrine model of hypertension. Taken together, these data suggest that mechanical factors may be responsible in part for the upregulation of expression. Consistent with this interpretation, we found that cultured rat aortic vascular smooth muscle cells exposed to mechanical strain (20% peak deformation at 1 Hz) exhibited a marked increase in MCP-1 expression, suggesting the hemodynamic strain imparted onto arterial cells in hypertension is an important stimulus underlying this phenomenon. These results provide important insights into the in vivo regulation of MCP-1 and have potential implications for understanding the influence of hypertension on atherosclerosis.

Key Words: angiotensin II • monocytes • macrophages • atherosclerosis • norepinephrine

	Introduction

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It has been proposed that hypertension, like atherosclerosis, is associated with a vascular inflammatory response that is characterized in part by recruitment of macrophages into the arterial wall.¹ An important mediator of monocyte recruitment into the vascular wall is the chemokine MCP-1. MCP-1 is secreted by vascular and circulating cells and induces undifferentiated monocyte migration to sites of active inflammation. Recent studies have detected increased MCP-1 levels in human atherosclerotic arteries² and experimental atherosclerotic lesions,^2,3 implying an important role for this molecule in the pathogenesis of vascular lesion formation.

We sought to determine the effect of hypertension on MCP-1 expression in the vascular wall. We analyzed MCP-1 mRNA levels in the aortas of rats made hypertensive by angiotensin II or norepinephrine infusion and compared these with levels in angiotensin II–infused rats treated with the nonspecific vascular smooth muscle relaxant hydralazine. To gain further mechanistic insight, we mechanically simulated the hypertensive environment in vitro by exposing cultured rat aortic smooth muscle cells to mechanical deformation and measured the effect of cyclic strain on MCP-1 mRNA expression.

	Methods

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Reagents and Materials
Losartan was a gift from Dr R.D. Smith (Dupont de Nemours Co, Wilmington, DE). TRI reagent was purchased from Molecular Research Center. Alzet osmotic minipumps were from Alza Corp. [³²P]dCTP was purchased from DuPont-New England Nuclear, and all rats were from Harlan Sprague-Dawley, Inc. Magna NT nylon membranes were from Micron Separation, Inc. Prime-it II probe labeling kits were purchased from Stratagene, and Biospin P30 columns were from BioRad. The mouse anti-rat macrophage antibody ED-1 was purchased from Accurate Chemical and Scientific Corp. Horse anti-mouse IgG was purchased from Vector Laboratories. All other chemicals and drugs were purchased from Sigma Chemical Co.

Experimental Models of Hypertension and Antihypertensive Therapy
Male Sprague-Dawley rats (weight, 250 to 300 g) received angiotensin II (0.75 mg · kg^-1 · d^-1) or norepinephrine (2.8 mg · kg^-1 · d^-1) infusions from implanted osmotic minipumps for 7 days (Alzet model 2002). Rats were anesthetized with 10 mg/kg xylazine IP and 80 mg/kg ketamine hydrochloride IP. For the angiotensin II–treated animals, an osmotic pump containing angiotensin II dissolved in a solution of 0.15 mol/L NaCl and 0.01 mol/L acetic acid was placed into the subcutaneous space. For the norepinephrine experiments, the pumps were connected to a polyethylene catheter placed in the internal jugular vein. Control animals were either sham-operated with no pump implanted or implanted with pumps containing vehicle only. The skin was closed with surgical staples. Systolic blood pressure was measured at baseline and before death with a tail-cuff sphygmomanometer.⁴ Measurements were done in triplicate, and a mean value was obtained.

Additional rats were treated with antihypertensive drugs. Losartan, a nonpeptide, competitive antagonist of the angiotensin II AT₁ receptor (25 mg · kg^-1 · d^-1), or the nonspecific vascular smooth muscle relaxant hydralazine (15 mg · kg^-1 · d^-1), was administered in the drinking water. Antihypertensive drug treatment was initiated 24 hours before pump implantation.

Tissue Harvesting and RNA Preparation
Sprague-Dawley rats were killed 7 days after pump implantation by CO₂ narcosis. A midline ventral incision was made, and with the heart still beating, an intracardiac injection of 5000 U heparin was administered. The aorta was then dissected from the renal arteries to the ascending arch, removed en bloc, and placed in ice-cold PBS, pH 7.4. Extravascular tissue was removed rapidly with forceps, and the vessel lumen was flushed sequentially with heparin and PBS. The specimen was placed in a precooled microcentrifuge tube, immediately submerged in liquid nitrogen, and transferred to a -70°C freezer for storage. Aortas were homogenized in TRI Reagent using a Polytron homogenizer (Brinkmann Instruments). RNA extraction was achieved using the TRI reagent method as described previously.⁵ Then, 10 µg total RNA per specimen was loaded onto a formaldehyde-containing 1% agarose gel.

Northern Blotting and Hybridization
After size-fractionation on a denaturing agarose-formaldehyde gel, total RNA was transferred to a nylon membrane (MSI, Inc) and exposed to ultraviolet light to cross-link the RNA. Membranes were prehybridized for 2 hours at 42°C with prehybridization buffer (50% [vol/vol] formamide, 1 mol/L NaCl, 5x Denhardt's solution, 0.5% sodium dodecyl sulfate, 50 mmol/L Tris buffer, pH 7.4, and 100 µg/mL denatured salmon testes DNA). Subsequently, membranes were hybridized overnight with a random-primed ³²P-dCTP-labeled rat cDNA probe. Membranes were then washed two to four times for 15 minutes in 0.5x standard saline citrate and 0.1% sodium dodecyl sulfate at 55°C and exposed to x-ray film at -70°C for 24 to 48 hours. Autoradiographic bands were quantified via densitometry and normalized to 28S ribosomal RNA.

Immunohistochemistry
Macrophages were identified with a mouse anti-rat macrophage antibody (ED-1) at a 1:250 dilution. Frozen paraformaldehyde-fixed tissue sections were thawed and fixed in acetone for 5 minutes, dried, and rehydrated in PBS. The primary antibody was applied at the indicated dilution in 1.0% BSA in PBS and incubated with a biotinylated secondary antibody (horse anti-mouse IgG at a 1:400 dilution) in PBS containing 1.0% BSA and 2.0% normal horse serum for 30 minutes at room temperature. This was followed by washing in PBS and incubation with the avidin-biotin enzyme complex and chromogenic substrate (Vector Red) as described by the manufacturer.

Stretch Experiments
VSMCs were isolated from the thoracic aortas of rats as described previously.⁶ Cells were grown in DMEM supplemented with 4500 mg/L D-glucose, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 25 mmol/L HEPES, and 10% heat-inactivated calf serum. Cells were plated onto flexible collagen-coated membranes (Flexcell International Corp) and fed every other day until near confluent. Wells were washed twice with serum-free DMEM and placed in 0.1% serum DMEM for 72 hours. The flexible membranes were stretched by vacuum using the Flexercell Strain Unit (model FX-2000).⁷ The degree of deformation of the membrane is a nonlinear function of the degree of negative pressure applied, and the strain gradient is a nonlinear function of radial, but not axial, position. A negative pressure of 13 kPa was applied beneath the membrane (radius, 12.7 mm) for all experiments, corresponding to a maximum deformation of 20% at 9.5 mm from the center of the well, decreasing exponentially toward the center and the periphery of the well. The strain unit was housed in an incubator at 5% CO₂ in humidified air at 37°C.

	Results

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Angiotensin II infusion resulted in a reproducible increase in systolic blood pressure from a baseline level of 130±3 to 208±9 mm Hg after 7 days of infusion (P<.001). Previous studies in our laboratory demonstrated that 7 days of angiotensin II infusion was required for the hypertensive response to fully develop.⁸ Fig 1 shows a photomicrograph of the thoracic aorta with immunostaining for monocytes/macrophages. Note that in addition to the marked medial hypertrophy, there is intense positive immunostaining with the ED-1 monoclonal antibody that is specific for rat monocytes and macrophages. Of interest is the predominance of the adventitial response. Although ED-1 positive cells were seen in the media, the most significant response appeared to involve the adventitia. There was no remarkable increase in the number of adherent monocytes on the endothelial surface.

View larger version (72K): [in this window] [in a new window]   Figure 1. Medial hypertrophy and identification of macrophages by immunohistochemistry in aortas of control and angiotensin II–treated animals. Sections were obtained from control animals (left) or animals treated with angiotensin II (0.75 mg · kg-1 · d-1) for 7 days (right). There is marked medial hyperplasia in the angiotensin II–treated animals. Macrophages were identified with the mouse anti-rat macrophage antibody ED-1. Aortas from sham-infused rats exhibited few scattered fibroblasts and no macrophages throughout the tunica adventitia. Numerous macrophages (red staining) were present in the aortic adventitia of hypertensive rats. Bar, 50 µm.

After 7 days of angiotensin II infusion, MCP-1 mRNA levels were measured with the use of quantitative Northern blot analysis. As can be seen in Fig 2, there was a significant 259±26% increase in MCP-1 mRNA expression in rats made hypertensive with angiotensin II compared with controls.

View larger version (27K): [in this window] [in a new window]   Figure 2. Increased expression of MCP-1 mRNA in the aortas of rats made hypertensive by angiotensin II (Ang II) infusion. Male Sprague-Dawley rats were treated with Ang II (0.75 mg · kg-1 · d-1) for 7 days. Some animals were treated with losartan (L). On day 7, the animals were killed, aortas were removed, and RNA was extracted with the use of TRI reagent. MCP-1 expression was determined by Northern blotting. Shown are representative blots and mean data (n=4 per group). *P<.01, compared with control.

To gain further mechanistic insights into the signaling mechanisms responsible for increased MCP-1 expression in angiotensin II–induced hypertension, we studied the effects of several different antihypertensive agents on MCP-1 expression in hypertensive rats. As can be seen in Fig 2, we found that losartan caused a complete inhibition of both the hypertensive response (107±10% inhibition) and the associated increase in MCP-1 expression (95±4% inhibition). These data are consistent with an AT₁ receptor–mediated process.

The nonselective vasodilator hydralazine also caused a significant, but incomplete, inhibition of increased MCP-1 mRNA expression in angiotensin II–treated rats (56±19% inhibition). This occurred despite complete normalization of the blood pressure response (98±9% inhibition; Fig 3). These data suggest that in addition to the direct humoral effects of angiotensin II, the resultant mechanical effects of blood pressure elevation may play an important role in the regulation of aortic MCP-1 expression.

View larger version (31K): [in this window] [in a new window]   Figure 3. Effect of antihypertensive treatment on MCP-1 mRNA expression in aortic tissue of hypertensive rats. Animals were prepared as above except that some animals were treated with hydralazine (H; 15 mg · kg-1 · d-1). Shown are representative blots and mean data (n=6 per group). Ang II indicates angiotensin II. *P<.01, compared with control. +P<.01, compared with Ang II.

To test this hypothesis, two different approaches were taken. First, we used another model of hypertension to exclude direct effects of angiotensin II. Using a norepinephrine model of hypertension, we found that there was a similar increase in aortic MCP-1 expression (Fig 4). Normalization of blood pressures with hydralazine completely eliminated the upregulation of MCP-1 expression. We also used an in vitro system to directly test the effects of cell deformation on MCP-1 expression. We exposed cultured rat aortic smooth muscle cells to a maximal 20% cyclic deformation at 1 Hz. Fig 5 shows a representative Northern blot depicting the time course of MCP-1 upregulation by mechanical deformation that demonstrates a rapid increase in MCP-1 expression.

View larger version (27K): [in this window] [in a new window]   Figure 4. Increased expression of MCP-1 mRNA in the aortas of rats made hypertensive by norepinephrine (NE) infusion. Male Sprague-Dawley rats were treated with NE (2.8 mg · kg-1 · d-1 IV) for 7 days. On day 7, the animals were killed, aortas were removed, and RNA was extracted using TRI reagent. MCP-1 expression was determined by Northern blotting. Shown are representative blots and mean data (n=4 per group). H indicates hydralazine. *P<.01, compared with control.

View larger version (78K): [in this window] [in a new window]   Figure 5. Effect of cyclic deformation on MCP-1 expression in VSMCs. VSMCs were plated onto collagen-coated, flexible-bottom tissue culture dishes and exposed to a maximal 20% cyclic deformation at 1 Hz for the indicated times. RNA extraction and Northern analysis were performed as above. Shown is a representative blot from a series of three such experiments.

	Discussion

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The present study demonstrates for the first time that the chemokine MCP-1 is upregulated in arteries of hypertensive rats. Antihypertensive drug therapy with hydralazine significantly reduced MCP-1 mRNA levels in an angiotensin II model of hypertension and completely eliminated the upregulation in a norepinephrine model of hypertension, suggesting that hemodynamic effects are important stimuli for MCP-1 regulation in vivo. Consistent with this finding, we demonstrated an upregulation of MCP-1 mRNA in cultured VSMCs exposed to pulsatile stretch. Taken together, these data demonstrate that arterial MCP-1 expression is upregulated in hypertension and that mechanical deformation of the arterial wall may, at least in part, be an important early signal for this phenomenon.

The results presented here may help to explain previous studies demonstrating an arterial inflammatory response in hypertension. Clozel et al⁹ have shown that in the cerebral vasculature of spontaneously hypertensive rats, monocyte/macrophage infiltration of the arterial wall occurs during the development of hypertension. Similar results have been seen in the Dahl salt-sensitive model of hypertension¹⁰ and in spontaneously hypertensive rats.¹¹ In this study, we have shown a marked inflammatory response of the arterial wall in hypertension that is characterized by marked infiltration of the adventitia with inflammatory cells. Our findings of upregulated MCP-1 expression provide a potential mechanism for the recruitment of monocytes. Additional mechanisms involving the upregulation of adhesion molecules are also likely to be involved in the development of the inflammatory response. The ultimate importance of a monocytic infiltration of the vascular wall in hypertension remains to be determined; however, monocytes/macrophages have been implicated in the regulation of vascular proliferation and vascular remodeling. The recruitment and differentiation of monocytes within the vascular wall may be a pivotal step in the development of both adaptive and maladaptive changes in hypertensive arteries.

The prominence of the monocyte/macrophage infiltration of the adventitia is indeed striking in that it resembles the response of the adventitia that is seen in arteritis, in atherosclerosis,¹² and after balloon injury.¹³ In atherosclerosis, the degree of adventitial infiltration corresponds with more advanced vascular lesions.¹² Although the presence of a cause-and-effect relationship between adventitial inflammation and intimal vascular disease in atherosclerosis has yet to be demonstrated, it has been shown that adventitial injury or removal results in endothelial cell loss and the subsequent development of neointimal hyperplasia.^14,15 We observed only occasional inflammatory cells that appeared to be adherent to the endothelial surface. Given the techniques used here, it would not be possible to accurately assess whether this was altered in the hypertensive animals.

Previous reports have documented an increased presence of MCP-1 in human and experimental atherosclerotic plaques,^2,3 with most of these studies identifying VSMCs and macrophages as the major sources.³ Cell culture studies have determined that MCP-1 accounts for a large portion of the monocyte chemoattractant activity produced by VSMCs.¹⁶ Thus, VSMC-derived MCP-1 is likely to play a major role in the early pathogenesis of vascular lesions and might logically be expected to be increased in disorders known to influence the development of atherosclerosis. Indeed, this has already been shown in a model of hypercholesterolemia.³ We have shown a significant upregulation of MCP-1 mRNA in the aortic walls of rats made hypertensive by angiotensin II. Because hypertension is a well known risk factor for the development of atherosclerosis in humans, the present study has implications for understanding this relationship on a fundamental level.

Considerable controversy exists regarding the relative importance of humoral versus hemodynamic stimuli as the predominant signal for the adverse effects of hypertension on arterial wall. Alderman et al¹⁷ found that humans with hypertension characterized by high circulating levels of renin and angiotensin II have three times the myocardial infarction risk of hypertensive patients without high renin levels, a relationship that persisted after controlling for conventional risk factors. Rajagopalan et al¹⁸ recently demonstrated that rats with angiotensin II–, but not catecholamine-, induced hypertension have impaired endothelium-dependent vasodilatation and increased vascular superoxide production. Both of these studies suggest that the blood pressure elevation induced by angiotensin II (ie, the mechanical stimulus) is not as important as other actions of angiotensin II in mediating the deleterious effects of hypertension on the vasculature. However, studies using aortic coarctation models of hypertension have shown that the arterial segment proximal to the coarctation, and thus subject to increased hemodynamic stress, is more prone vascular hypertrophy than the low-pressure segment below the stenosis,¹⁹ suggesting a more important role for hemodynamic forces in the vascular pathology associated with high renin hypertension. In the present study, we reduced blood pressure in angiotensin II–infused rats using either the specific angiotensin II receptor antagonist losartan or the nonspecific vasodilator hydralazine. This enabled us to compare the effects of blood pressure lowering via angiotensin II antagonism with vasorelaxation in the presence of persistent angiotensin II signaling. Both strategies were effective in inhibiting MCP-1 induction, although the inhibition by hydralazine was incomplete despite normalization of blood pressure. Therefore, angiotensin II may have a direct effect on MCP-1 expression, as suggested by others.²⁰ In the norepinephrine-treated animals, we found that not only did norepinephrine cause a similar increase in MCP-1 expression but also normalization of the blood pressure with hydralazine completely inhibited the increased MCP-1 expression. Thus, in regard to MCP-1 mRNA regulation in hypertension, we can conclude that both humoral and mechanical mechanisms appear to be involved and that in the specific case of angiotensin II–induced hypertension, elevated blood pressure and its consequences appear to be as important as the nonpressor effects of angiotensin II.

Hydralazine may have chemical properties in addition to its vasodilator capacity. Munzel et al²¹ recently demonstrated that hydralazine is capable of inhibiting membrane NADH oxidase activity in arterial segments. Thus, it is possible that administration of hydralazine in these experiments was in effect administration of an antioxidant. This is potentially important because aortic membrane–associated NADH oxidase is upregulated in angiotensin II–induced hypertension²² and MCP-1 expression is upregulated by reactive oxygen species.²³ However, our experiments showing a direct upregulation of MCP-1 expression by mechanical strain and our studies with the norepinephrine-treated rats that do not produce excess superoxide²⁴ argue against this possibility.

A cardinal feature of hypertension is increased pressure and strain on the arterial wall, which is transmitted to endothelial and smooth muscle cells. Recent advances in cell biology have allowed investigators to study cells cultured in mechanical environments that more closely mimic the in vivo situation.^7,25 Cyclic strain, a force experienced by cells in the arterial wall, induced a brisk upregulation of MCP-1 mRNA that was sustained for 4 hours. This finding is consistent with our in vivo experiments and implicates mechanical forces in the upregulation of this chemokine.

In summary, hypertension is associated with a marked inflammatory response and an upregulation of MCP-1 mRNA in the aortic wall. Elevated blood pressure, and the resultant mechanical strain on the aortic smooth muscle cells, appears to be as important as direct angiotensin II signaling in the upregulation of MCP-1, thus underscoring the importance of hemodynamic forces in angiotensin II–induced hypertension. MCP-1 may subsequently play a central role in the development of hypertensive vascular disease through its effect on the recruitment of monocytes into the vascular wall. In addition, the known association between MCP-1 and atherosclerotic lesions provides potential mechanistic insights into the molecular basis for hypertension as a risk factor for the development of atherosclerosis.

	Selected Abbreviations and Acronyms

 

BSA = bovine serum albumin DMEM = Dulbecco's modified Eagle's medium MCP-1 = monocyte chemoattractant protein-1 PBS = phosphate-buffered saline VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by a VA Merit Review Board grant (Dr Taylor) and NIH NRSA grant HL-09185 (Dr Capers).

Received July 9, 1997; first decision July 15, 1997; accepted July 15, 1997.

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				S. G. Lakoski, M. Cushman, W. Palmas, R. Blumenthal, R. B. D'Agostino Jr, and D. M. Herrington The Relationship Between Blood Pressure and C-Reactive Protein in the Multi-Ethnic Study of Atherosclerosis (MESA) J. Am. Coll. Cardiol., November 15, 2005; 46(10): 1869 - 1874. [Abstract] [Full Text] [PDF]

				N. Ishizaka, K. Saito, I. Mori, G. Matsuzaki, M. Ohno, and R. Nagai Iron Chelation Suppresses Ferritin Upregulation and Attenuates Vascular Dysfunction in the Aorta of Angiotensin II-Infused Rats Arterioscler. Thromb. Vasc. Biol., November 1, 2005; 25(11): 2282 - 2288. [Abstract] [Full Text] [PDF]

				D. M. Shah Role of the renin-angiotensin system in the pathogenesis of preeclampsia Am J Physiol Renal Physiol, April 1, 2005; 288(4): F614 - F625. [Abstract] [Full Text] [PDF]

				G. Gruden, G. Setti, A. Hayward, D. Sugden, S. Duggan, D. Burt, R. E. Buckingham, L. Gnudi, and G. Viberti Mechanical Stretch Induces Monocyte Chemoattractant Activity via an NF-{kappa}B-Dependent Monocyte Chemoattractant Protein-1-Mediated Pathway in Human Mesangial Cells: Inhibition by Rosiglitazone J. Am. Soc. Nephrol., March 1, 2005; 16(3): 688 - 696. [Abstract] [Full Text] [PDF]

				H. M. Dourron, G. M. Jacobson, J. L. Park, J. Liu, D. J. Reddy, M. L. Scheel, and P. J. Pagano Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H946 - H953. [Abstract] [Full Text] [PDF]

				J. Liu, A. Ormsby, N. Oja-Tebbe, and P. J. Pagano Gene Transfer of NAD(P)H Oxidase Inhibitor to the Vascular Adventitia Attenuates Medial Smooth Muscle Hypertrophy Circ. Res., September 17, 2004; 95(6): 587 - 594. [Abstract] [Full Text] [PDF]

				M. Ishibashi, K.-i. Hiasa, Q. Zhao, S. Inoue, K. Ohtani, S. Kitamoto, M. Tsuchihashi, T. Sugaya, I. F. Charo, S. Kura, et al. Critical Role of Monocyte Chemoattractant Protein-1 Receptor CCR2 on Monocytes in Hypertension-Induced Vascular Inflammation and Remodeling Circ. Res., May 14, 2004; 94(9): 1203 - 1210. [Abstract] [Full Text] [PDF]

				F. Kuwahara, H. Kai, K. Tokuda, M. Takeya, A. Takeshita, K. Egashira, and T. Imaizumi Hypertensive Myocardial Fibrosis and Diastolic Dysfunction: Another Model of Inflammation? Hypertension, April 1, 2004; 43(4): 739 - 745. [Abstract] [Full Text] [PDF]

				W. Ni, S. Kitamoto, M. Ishibashi, M. Usui, S. Inoue, K.-i. Hiasa, Q. Zhao, K.-i. Nishida, A. Takeshita, and K. Egashira Monocyte Chemoattractant Protein-1 Is an Essential Inflammatory Mediator in Angiotensin II-Induced Progression of Established Atherosclerosis in Hypercholesterolemic Mice Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 534 - 539. [Abstract] [Full Text]

				K. Tokuda, H. Kai, F. Kuwahara, H. Yasukawa, N. Tahara, H. Kudo, K. Takemiya, M. Koga, T. Yamamoto, and T. Imaizumi Pressure-Independent Effects of Angiotensin II on Hypertensive Myocardial Fibrosis Hypertension, February 1, 2004; 43(2): 499 - 503. [Abstract] [Full Text] [PDF]

				G. J. Blake, N. Rifai, J. E. Buring, and P. M Ridker Blood Pressure, C-Reactive Protein, and Risk of Future Cardiovascular Events Circulation, December 16, 2003; 108(24): 2993 - 2999. [Abstract] [Full Text] [PDF]

				S. Hayashidani, H. Tsutsui, T. Shiomi, M. Ikeuchi, H. Matsusaka, N. Suematsu, J. Wen, K. Egashira, and A. Takeshita Anti-Monocyte Chemoattractant Protein-1 Gene Therapy Attenuates Left Ventricular Remodeling and Failure After Experimental Myocardial Infarction Circulation, October 28, 2003; 108(17): 2134 - 2140. [Abstract] [Full Text] [PDF]

				M. Higashi, H. Shimokawa, T. Hattori, J. Hiroki, Y. Mukai, K. Morikawa, T. Ichiki, S. Takahashi, and A. Takeshita Long-Term Inhibition of Rho-Kinase Suppresses Angiotensin II-Induced Cardiovascular Hypertrophy in Rats In Vivo: Effect on Endothelial NAD(P)H Oxidase System Circ. Res., October 17, 2003; 93(8): 767 - 775. [Abstract] [Full Text] [PDF]

				J. Liu, F. Yang, X.-P. Yang, M. Jankowski, and P. J. Pagano NAD(P)H Oxidase Mediates Angiotensin II-Induced Vascular Macrophage Infiltration and Medial Hypertrophy Arterioscler. Thromb. Vasc. Biol., May 1, 2003; 23(5): 776 - 782. [Abstract] [Full Text] [PDF]