Vascular smooth muscle cell mechanotransduction through serum and glucocorticoid inducible kinase-1 promotes interleukin-6 production and macrophage accumulation in murine hypertension

by Selleckchem | Dec 27 2023

Objective: The objective of this investigation was to demonstrate that in vivo induction of hypertension (HTN) and in vitro cyclic stretch of aortic vascular smooth muscle cells (VSMCs) can cause serum and glucocorticoid-inducible kinase (SGK-1)-dependent production of cytokines to promote macrophage accumulation that may promote vascular pathology.

Methods: HTN was induced in C57Bl/6 mice with angiotensin II infusion (1.46 mg/kg/day × 21 days) with or without systemic infusion of EMD638683 (2.5 mg/kg/day × 21 days), a selective SGK-1 inhibitor. Systolic blood pressure was recorded. Abdominal aortas were harvested to quantify SGK-1 activity (pSGK-1/SGK-1) by immunoblot. Flow cytometry quantified the abundance of CD11b+/F480+ cells (macrophages). Plasma interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was assessed by enzyme-linked immunosorbent assay. Aortic VSMCs from wild-type mice were subjected to 12% biaxial cyclic stretch (Stretch) for 3 or 12 hours with or without EMD638683 (10 μM) and with or without SGK-1 small interfering RNA with subsequent quantitative polymerase chain reaction for IL-6 and MCP-1 expression. IL-6 and MCP-1 in culture media were analyzed by enzyme-linked immunosorbent assay. Aortic VSMCs from SGK-1flox+/+ mice were transfected with Cre-Adenovirus to knockdown SGK-1 (SGK-1KD VSMCs) and underwent parallel tension experimentation. Computational modeling was used to simulate VSMC signaling. Statistical analysis included analysis of variance with significance at a P value of <.05.

Results: SGK-1 activity, abundance of CD11b+/F4-80+ cells, and plasma IL-6 were increased in the abdominal aorta of mice with HTN and significantly reduced by treatment with EMD638683. This outcome mirrored the increased abundance of IL-6 in media from Stretch C57Bl/6 VSMCs and attenuation of the effect with EMD638683 or SGK-1 small interfering RNA. C57Bl/6 VSMCs also responded to Stretch with increased MCP-1 expression and secretion into the culture media. Further supporting the integral role of mechanical signaling through SGK-1, target gene expression and cytokine secretion was unchanged in SGK-1KD VSMCs with Stretch, and computer modeling confirmed SGK-1 as an intersecting node of signaling owing to mechanical strain and angiotensin II.

Conclusions: Mechanical activation of SGK-1 in aortic VSMCs can promote inflammatory signaling and increased macrophage abundance, therefore this kinase warrants further exploration as a pharmacotherapeutic target to abrogate hypertensive vascular pathology.

Comments:

It seems like your investigation aimed to explore the role of serum and glucocorticoid-inducible kinase-1 (SGK-1) in the development of vascular pathology in hypertension (HTN). Here's a breakdown of your methods and results:

### Methods:
1. **Induction of HTN in mice:** Angiotensin II infusion was used to induce HTN in C57Bl/6 mice.
2. **SGK-1 inhibition:** EMD638683, a selective SGK-1 inhibitor, was administered to some mice undergoing HTN induction.
3. **Measurement techniques:**
   - Systolic blood pressure recording.
   - Assessment of SGK-1 activity in the abdominal aortas via immunoblot.
   - Flow cytometry to quantify macrophage abundance.
   - Measurement of plasma interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) levels.
4. **In vitro experiments with aortic vascular smooth muscle cells (VSMCs):**
   - Subjecting VSMCs to cyclic stretch to simulate mechanical strain.
   - Examining IL-6 and MCP-1 expression and secretion in response to stretch.
   - Using SGK-1 small interfering RNA and inhibitors to modulate SGK-1 activity in VSMCs.
5. **Genetic manipulation:**
   - Knocking down SGK-1 in VSMCs using Cre-Adenovirus in SGK-1flox+/+ mice.
6. **Computational modeling:** Simulating VSMC signaling to understand the role of SGK-1 in response to mechanical strain and angiotensin II.

### Results:
1. **Increased SGK-1 activity:** HTN induction led to elevated SGK-1 activity in the aorta, along with increased macrophage abundance and plasma IL-6 levels.
2. **SGK-1 inhibition effects:** Treatment with EMD638683 reduced SGK-1 activity, macrophage abundance, and IL-6 levels, suggesting a potential role for SGK-1 in the inflammatory response.
3. **In vitro findings:** Stretching VSMCs led to increased IL-6 and MCP-1 expression and secretion, which was mitigated by SGK-1 inhibition or knockdown.
4. **SGK-1 knockdown:** VSMCs with reduced SGK-1 did not exhibit changes in gene expression or cytokine secretion in response to stretch.
5. **Computational modeling:** Confirmed SGK-1 as a crucial signaling node in response to mechanical strain and angiotensin II.

### Conclusions:
The study suggests that mechanical activation of SGK-1 in aortic VSMCs might drive inflammatory signaling, leading to increased macrophage accumulation in hypertensive conditions. As such, targeting SGK-1 could potentially serve as a therapeutic approach to mitigate hypertensive vascular pathology.

This investigation provides valuable insights into the molecular mechanisms underlying hypertension-induced vascular pathology and highlights SGK-1 as a potential therapeutic target.