Recruitment of macrophages from the spleen contributes to myocardial fibrosis and hypertension induced by angiotensin II

Abstract

Introduction: The purpose of this study was to determine whether macrophages migrated from the spleen are associated with angiotensin II-induced cardiac fibrosis and hypertension.

Methods: Sprague-Dawley rats were subjected to angiotensin II infusion in vehicle (500 ng/kg/min) for up to four weeks. In splenectomy, the spleen was removed before angiotensin II infusion. In the angiotensin II AT1 receptor blockade, telmisartan was administered by gastric gavage (10 mg/kg/day) during angiotensin II infusion. The heart and aorta were isolated for Western blot analysis and immunohistochemistry.

Results: Angiotensin II infusion caused a significant reduction in the number of monocytes in the spleen through the AT1 receptor-activated monocyte chemoattractant protein-1. Comparison of angiotensin II infusion, splenectomy and telmisartan comparatively reduced the recruitment of macrophages into the heart. Associated with this change, transforming growth factor β1 expression and myofibroblast proliferation were inhibited, and Smad2/3 and collagen I/III were downregulated. Furthermore, interstitial/perivascular fibrosis was attenuated. These modifications occurred in coincidence with reduced blood pressure. At week 4, invasion of macrophages and myofibroblasts in the thoracic aorta was attenuated and expression of endothelial nitric oxide synthase was upregulated, along with a reduction in aortic fibrosis.

Conclusions: These results suggest that macrophages when recruited into the heart and aorta from the spleen potentially contribute to angiotensin II-induced cardiac fibrosis and hypertension.

Keywords: Angiotensin II AT1 receptor; collagen; hypertension; macrophages; myocardial fibrosis; splenectomy.

Figure 1.

Detection of monocytes and expression of the AT1 receptor from paraffin-embedded transverse sections in normal rat spleen. The low-power view in (a) shows the average amount of two types of tissue: the red pulp and white pulp in the parenchyma of the spleen. The red pulp makes up the majority of the spleen and serves as an important reservoir for large quantities of phagocytic white blood cells called monocytes. White pulp is lymphatic tissue that mainly consists of lymphocytes called B-lymphocytes and T-lymphocytes that surround arteries. Venous sinuses are essentially cavities filled with blood. The presence of macrophages and expression of the AT1 receptor were confirmed by immunohistochemistry (b). In a higher magnification of the cross-section, it was found that monocytes are predominantly located within the red pulp, where the positively stained AT1 receptor shown as brown granular pigment was identified. Magnification×400; scale bars: 100 µm (n=6).

Figure 2.

Angiotensin II (Ang II) receptor expression, macrophage accumulation and monocyte chemoattractant protein-1 (MCP-1) level in the spleen during Ang II infusion. Protein levels of the AT1/AT2 receptors (a) and MCP-1 (d) were analyzed using Western blot and all bands were normalized by actin. Expression of the AT1 receptor (b) and macrophage accumulation (c) were detected with immunohistochemistry. Macrophage localization was determined by the number of positively-stained cells (brown granular pigment) in each high-powered field (HPF; original magnification: ×400; scale bars: 100 µm). Sham: rats were infused with a saline pump for four weeks; Ang II: rats received Ang II subcutaneous infusion at a rate of 500 ng/kg/min; Telmi: telmisartan plus Ang II. Telmisartan was administered by gastric gavage at a dose of 10 mg/kg/day during Ang II infusion. Values are means±standard error of the mean (SEM) (n=7/each group). ^*p<0.05 Ang II vs Sham; ^†p<0.05 Telmi vs Ang II.

Figure 3.

Accumulation of macrophages, protein level of transforming growth factor β1 (TGFβ1) and proliferation of myofibroblasts in the heart during angiotensin II (Ang II) infusion. The presence of macrophages (a) and myofibroblasts (c) and expression of TGFβ1 (b) were detected using immunohistochemistry and Western blot. The number of macrophages and myofibroblast was determined by positively-stained cells (brown and red granular pigments) in each high-powered field (HPF; original magnification: ×400; scale bars: 100 µm). Sham: rats were infused with a saline pump for four weeks; Ang II: rats received Ang II subcutaneous infusion at a rate of 500 ng/kg/min; Splen: splenectomy was performed before Ang II infusion; Telmi: telmisartan was administered orally during Ang II infusion; S+T: splenectomy was performed and telmisartan was administered during Ang II infusion. Values are means±standard error of the mean (SEM) (n=7/each group). ^*p<0.05 Ang II vs Sham; ^†p<0.05 Telmi vs Ang II.

Figure 4.

Protein levels of pSmad2, pSmad3, collagen I and III in the heart during angiotensin II (Ang II) infusion. Expression of Smads and collagens were analyzed using Western blot. All bands were normalized by actin as illustrated among groups. Sham: rats were infused with a saline pump for four weeks; Ang II: rats received Ang II subcutaneous infusion at a rate of 500 ng/kg/min; Splen: splenectomy was performed before Ang II infusion; Telmi: telmisartan was administered orally during Ang II infusion; S+T: splenectomy was performed and telmisartan was administered during Ang II infusion. Values are means±standard error of the mean (SEM) (n=7/each group). ^*p<0.05 Ang II vs Sham; ^†p<0.05 Telmi vs Ang II.

Figure 5.

Representative photomicrographs of myocardial cross sections stained with Masson’s trichrome method. Interstitial fibrosis (a) and perivascular fibrosis (b) at week 4 of angiotensin II (Ang II) infusion were identified by blue staining. Interstitial and perivascular fibrosis were calculated as percent fibrotic area and fibrosis ratio (PFR) in each high-powered field (HPF; original magnification: ×400; scale bars: 100 µm). Sham: rats were infused with a saline pump for four weeks; Ang II: rats received Ang II subcutaneous infusion at a rate of 500 ng/kg/min; Splen: splenectomy was performed before Ang II infusion; Telmi: telmisartan was administered orally during Ang II infusion; S+T: splenectomy was performed and telmisartan was administered during Ang II infusion. Values are means±standard error of the mean (SEM) (n=7/each group). ^*p<0.05 Ang II vs Sham; ^†p<0.05 Telmi vs Ang II.

Figure 6.

Change in blood pressure during angiotensin II (Ang II) infusion. Blood pressure was measured by inflating a tail cuff with simultaneous monitoring of the cuff pressure. The arrows indicate systolic blood pressure (SBP) and diastolic blood pressure (DBP). Change in blood pressure among groups can be identified by the distance during cuff inflation between the baseline (Base) and the beginning of SBP tracing. Sham: rats were infused with a saline pump for four weeks; Ang II: rats received Ang II subcutaneous infusion at a rate of 500 ng/kg/min; Splen: splenectomy was performed before Ang II infusion; Telmi: telmisartan was administered orally during Ang II infusion; S+T: splenectomy was performed and telmisartan was administered during Ang II infusion. Values are means± standard error of the mean (SEM) (n=7/each group). ^*p<0.05 Ang II vs Sham; ^†p<0.05 Telmi vs Ang II.

Figure 7.

Representative photomicrographs of aortic cross sections stained with immunohistochemistry and Masson’s trichrome method. In each high-powered field (HPF; original magnification: ×400; scale bars: 100 µm), the recruitment of macrophages (MAC; (a)) and myofibroblasts (MYOFIB, (b)) in aortic intima and adventitia was determined by the number of positively-stained cells (brown and red granular pigments as indicated by arrows). Expression of eNOS (c) and the AT1 receptor (d) was identified by brown staining in the aortic intima and media. Collagen deposition in aorta (e) was analyzed by the thickness of adventitial fibrotic area shown as blue staining. Sham: rats were infused with a saline pump for four weeks; Ang II: rats received Ang II subcutaneous infusion at a rate of 500 ng/kg/min; Splen: splenectomy was performed before Ang II infusion; Telmi: telmisartan was administered orally during Ang II infusion; Splen+Telmi: splenectomy was performed and telmisartan was administered during Ang II infusion; n=6/each group.