J Bacteriol Virol.  2015 Jun;45(2):138-150. 10.4167/jbv.2015.45.2.138.

CCL5 Inhibits Elevation of Blood Pressure and Expression of Hypertensive Mediators in Developing Hypertension State Spontaneously Hypertensive Rats

• Kim HY
• Cha HJ
• Choi JH
• Kang YJ
• Park SY
• Kim HS

• KMID: 2149214
• DOI: http://doi.org/10.4167/jbv.2015.45.2.138

Abstract

CCL5, a proinflammatory chemokine, has been shown to attenuate angiotensin (Ang) II-induced expression of hypertensive mediators as well as Ang II-induced inhibition of anti-hypertensive mediator expression in vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHR). In the present study, functional roles of CCL5 on hypertension were examined in developing hypertension state SHR (DHSHR). DHSHR at an age of 8 weeks were injected CCL5 (1.5 microg/kg) subcutaneously twice a day for 3 weeks (SHRi, n=5). Control groups consisted of normal age-matched saline-treated SHR (SHRc, n=5) and normotensive Wistar-Kyoto rats (WKY, n=5). Effect of CCL5 on blood pressure was measured before treatment, weekly during treatment, and 1 day after the final injection. After injecting for 3 weeks, effects of CCL5 on expression of hypertensive mediators were examined in thoracic aorta tissues and VSMCs. Blood pressure in SHRi was maintained without any elevation during the treatment period, whereas blood pressure in SHRc progressively increased with age. Expression of Ang II subtype I receptor was reduced in SHRi thoracic aorta tissues and VSMCs compared to those in SHRc. In addition, expression levels of hypertensive mediators were significantly reduced in SHRi thoracic aorta tissues and VSMCs compared to those in SHRc. In contrast, AMP-activated protein kinase (AMPK) activity and interleukin-10 (IL-10) expression were elevated in SHRi thoracic aorta tissues and VSMCs compared to levels in SHRc. These results suggest that reduction of hypertensive mediators and elevation of anti-hypertensive mediators by CCL5 treatment promotes maintenance of blood pressure in DHSHR.

Keyword

CCL5; Hypertensive mediator; Vascular smooth muscle cells; Blood pressure

MeSH Terms

AMP-Activated Protein Kinases
Angiotensins
Animals
Aorta, Thoracic
Blood Pressure*
Hypertension*
Interleukin-10
Muscle, Smooth, Vascular
Rats
Rats, Inbred SHR*
AMP-Activated Protein Kinases
Angiotensins
Interleukin-10

Figure

• 

  Figure 1. CCL5 treatment inhibits elevation of blood pressure but has no effect on thoracic aorta wall thickness in DHSHR. (A) Body weight and blood pressure were measured before treatment, every week during treatment, and 1 day after final injection with CCL5 (1.5 μg/ kg) or normal saline twice per day for 3 weeks. Results are presented as means ± SEM (n=5 for each group). ∗ p < 0.05 vs. SHRc. ∗∗∗ p <0.001 vs. SHRc. (B) Aortic tissues were stained using H&E (upper: 40× magnification, lower: 200× magnification). WKY: WKY injected with normal saline, SHRc: DHSHR injected with normal saline, SHRi: DHSHR injected with CCL5.

• 

  Figure 2. CCL5 modulates expression of Ang II receptors in DHSHR thoracic aorta tissues and VSMCs. (A) Thoracic aorta tissues from each rat group were isolated and immunohistochemically labeled for AT1 R and AT2 R (200× magnification). (B, C) Total RNAs were isolated from thoracic aorta tissues and VSMCs of each rat group, and real-time PCR was performed. Bars are expressed as means ± SEM (n=5 for each group). ∗p < 0.05 vs. SHRc VSMCs. ∗∗p < 0.01 vs. SHRc thoracic aorta tissues. (D) Cell lysates were prepared from VSMCs from thoracic aorta tissues of each rat group, immunoblotting was performed. Each blot is representative of 4 silmilar experiments. ∗p <0.05 vs. SHRc VSMCs. WKY: WKY injected with normal saline, SHRc: DHSHR injected with normal saline, SHRi: DHSHR injected with CCL5.

• 

  Figure 3. CCL5 modulates expression of CCL2, CXCL8, and IL-10 in DHSHR thoracic aorta tissues and VSMCs. (A) Aortic cross sections were stained immunohistochemically for CCL2, CXCL8, and IL-10 (200× magnification). (B, C) Total RNAs were isolated from thoracic aorta tissues and VSMCs of each rat group, and real-time PCR was performed. Bars are expressed as means ± SEM (n=5 for each group). ∗p < 0.05 vs. SHRc thoracic aorta tissues. ∗p < 0.05 vs. SHRc VSMCs. WKY: WKY injected with normal saline, SHRc: DHSHR injected with normal saline, SHRi: DHSHR injected with CCL5.

• 

  Figure 4. Treatment of CCL5 reduces 12-LO and ET-1 expression in DHSHR thoracic aorta tissues and VSMCs. (A, B) After total RNA was isolated from thoracic aorta tissues and VSMCs, real-time PCR was performed. Bars are expressed as means ± SEM (n=5 for each group). ∗p < 0.05 vs. SHRc thoracic aorta tissues. ∗∗p < 0.01 vs. SHRc thoracic aorta tissues. ∗p < 0.05 vs. SHRc VSMCs. (C) Cell lysates were prepared from VSMCs of each rat group, and immunoblotting were performed. Each blot is representative of 3 silmilar experiments.∗ p < 0.05 vs. SHRc VSMCs. WKY: WKY injected with normal saline, SHRc: DHSHR injected with normal saline, SHRi: DHSHR injected with CCL5.

• 

  Figure 5. Treatment of CCL5 elevates activity of AMPK but has no effect on DDAH activity in DHSHR thoracic aorta tissues and VSMCs. (A) Total RNAs for real-time PCR was isolated from thoracic aorta tissues of each group. Additionally, cell lysates for immunoblotting was prepared from VSMCs from thoracic aorta tissues of each group. Each blot is representative of 3 silmilar experiments. ∗ p <0.05 vs. SHRc thoracic aorta tissues. a p < 0.05 vs. SHRc VSMCs. (B) Total RNAs were isolated from VSMCs and thoracic aorta tissues of each rat group, and real-time PCR was performed. In addition, DDAH activity was evaluated in VSMCs from thoracic aorta tissues of each rat group, Bars are expressed as means ± SEM (n=5 for each group). WKY: WKY injected with normal saline, SHRc: DHSHR injected with normal saline, SHRi: DHSHR injected with CCL5.

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