Inflammation and mechanical stretch promote aortic stiffening in hypertension through activation of p38 mitogen-activated protein kinase

Abstract

Rationale: Aortic stiffening commonly occurs in hypertension and further elevates systolic pressure. Hypertension is also associated with vascular inflammation and increased mechanical stretch. The interplay between inflammation, mechanical stretch, and aortic stiffening in hypertension remains undefined.

Objective: Our aim was to determine the role of inflammation and mechanical stretch in aortic stiffening.

Methods and results: Chronic angiotensin II infusion caused marked aortic adventitial collagen deposition, as quantified by Masson trichrome blue staining and biochemically by hydroxyproline content, in wild-type but not in recombination activating gene-1-deficient mice. Aortic compliance, defined by ex vivo measurements of stress-strain curves, was reduced by chronic angiotensin II infusion in wild-type mice (P<0.01) but not in recombination activating gene-1-deficient mice (P<0.05). Adoptive transfer of T-cells to recombination activating gene-1-deficient mice restored aortic collagen deposition and stiffness to values observed in wild-type mice. Mice lacking the T-cell-derived cytokine interleukin 17a were also protected against aortic stiffening. In additional studies, we found that blood pressure normalization by treatment with hydralazine and hydrochlorothiazide prevented angiotensin II-induced vascular T-cell infiltration, aortic stiffening, and collagen deposition. Finally, we found that mechanical stretch induces the expression of collagen 1α1, 3α1, and 5a1 in cultured aortic fibroblasts in a p38 mitogen-activated protein kinase-dependent fashion, and that inhibition of p38 prevented angiotensin II-induced aortic stiffening in vivo. Interleukin 17a also induced collagen 3a1 expression via the activation of p38 mitogen-activated protein kinase.

Conclusions: Our data define a pathway in which inflammation and mechanical stretch lead to vascular inflammation that promotes collagen deposition. The resultant increase in aortic stiffness likely further worsens systolic hypertension and its attendant end-organ damage.

Keywords: inflammation.

Figure 1. Role of T cells in aortic collagen deposition in hypertension

A, Effects of angiotensin II-induced hypertension on vascular collagen and elastin in WT and RAG-1^−/− mice. Perfusion-fixed sections of the thoracic aortas were sectioned (6 mm) and stained with Hematoxylin and Eosin, Masson’s trichome and Van Gieson’s Elastica staining to highlight collagen (blue) and elastin (black). Scale bar indicates 100 μm. B, Adventitial collagen area was quantified by planimetry. C, Aortic collagen quantification by hydroxyproline assay. Isolated thoracic aortas were digested in 6 N HCl at 105 °C for 48 hours before hydroxyproline was quantified. D, Elastin was quantified biochemically by initial separation in 0.1 N NaOH at 90 °C for 30 min and subsequently by ninhydrin assay. Ang II, angiotensin II. Data were analyzed using two-way ANOVA, n=6–9.

Figure 2. T cells mediate angiotensin II-induced aortic stiffening

Freshly-isolated aortas were mounted on a myograph system in Ca²⁺-free buffer to determine pressure-diameter relationships. A–D, compliance curves and stress-strain relationships were constructed from inner diameter and outer diameter. E and F, inner and outer diameter of WT and RAG-1^−/− mice measured at 25 mmHg step changes in pressure from 0–200 mmHg. Ang II, angiotensin II. Data were analyzed using one-way ANOVA with repeated measures, n=6–9.

Figure 3. Role of IL-17a in aortic stiffening

IL-17a deficient mice were infused with chronic angiotensin II (490ng/kg/min) for 14 days. A and B, the thoracic aortas were fix-perfused for Masson’s trichrome staining and quantified for adventitia collagen staining. Scale bar indicates 100 μm. C, segments of the thoracic aorta were digested in 6N HCl at 105 °C for 48 hours and measured for hydroxyproline concentration. Data analyzed using two-way ANOVA. D and E, compliance curves and stress-strain curves for IL-17a^−/− mice. Ang II, angiotensin II. Data analyzed using one-way ANOVA with repeated measures, n=6–8.

Figure 4. Antihypertensive treatment (antiHBP) prevents collagen deposition, aortic stiffening and T cell infiltration

Mice were treated with hydralazine and hydrochlorothiazide (320 mg/L and 60 mg/L in the drinking water) concurrently with angiotensin II infusion. Hyd, Hydralazine, HCTZ, hydrochlorothiazide. A and B, telemetry recording of blood pressures. D, day; N, night. The effects of blood pressure normalization on collagen depostion (C–E), aortic stiffening (F and G) and aorfic inflammatory cell infiltration (H–L) are shown. Scale bar indicates 100 μm. Ang II, angiotensin II. Data for blood pressure and aortic stiffness were analyzed using one-way ANOVA with repeated measurements (n=8). Collagen deposition and flow cytometry were analyzed using one-way ANOVA (n=8).

Figure 5. Effect of cyclical stretch and IL-17a on aortic fibroblasts and role of p38 MAP kinase

Mouse aortic fibrobasts were exposed to various degrees of cyclical stretch or the T cell cytokine IL-17a (100 ng/mL) in culture for 36 hours. A–C, both hypertensive mechanical stretch and IL-17a activated p38 MAPK in cultured mouse aortic fibroblasts. D–F, The effects of p38 inhibitor SB203580 (10 ng/mL) on mechanical stretch-induced expression of collagen subtypes in fibroblasts. G–I, The effects of p38 inhibition on IL-17a-induced collagen expression. Data analyzed using one-way ANOVA, n=3–6.

Figure 6. P38 MAP kinase mediates collagen deposition and aortic stiffening in angiotensin II-induced hypertension

A–C, Normalization of blood pressure prevented the activation of p38 MAP kinase in angiotensin II-infused mouse aortas. Data analyzed with one-way ANOVA (n=4–6). D and E, Effect of SB203580 on blood pressure. D, day; N, night. F–H, Effect of SB203580 on aortic collagen deposition. Data analyzed using one-way ANOVA (n=6–8). Mice received intraperitoneal injections of SB203580 (10 mg/kg/day) during angiotensin II infusion. Scale bar indicates 100 μm. I and J, Effect of SB203580 on aortic stiffening. Ang II, angiotensin II. Blood pressure and aortic stiffness were analyzed using one-way ANOVA with repeated measures (n=6–8).

Figure 7. Pathway showing interactions of mechanical stretch and inflammation in aortic stiffening

Increased mechanical stretch promotes perivascular T cell infiltration and activates p38 MAP kinase, leading to adventitial collagen deposition. This reduces aortic compliance and ultimately leads to loss of windkessel effect in large capacitance arteries such as the aorta.