Mohamed A. Saleh

DC isoketal-modified proteins activate T cells and promote hypertension

Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive γ-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs. Isoketal accumulation was associated with DC production of IL-6, IL-1β, and IL-23 and an increase in costimulatory proteins CD80 and CD86. These activated DCs promoted T cell, particularly CD8+ T cell, proliferation; production of IFN-γ and IL-17A; and hypertension. Moreover, isoketal scavengers prevented these hypertension-associated events. Plasma F2-isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension. Isoketal-modified proteins were also markedly elevated in circulating monocytes and DCs from humans with hypertension. Our data reveal that hypertension activates DCs, in large part by promoting the formation of isoketals, and suggest that reducing isoketals has potential as a treatment strategy for this disease.

Inflammation and Mechanical Stretch Promote Aortic Stiffening in Hypertension Through Activation of p38 Mitogen-Activated Protein Kinase

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 1&agr;1, 3&agr;1, 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.

Oligoclonal CD8+ T Cells Play a Critical Role in the Development of Hypertension

Recent studies have emphasized a role of adaptive immunity, and particularly T cells, in the genesis of hypertension. We sought to determine the T-cell subtypes that contribute to hypertension and renal inflammation in angiotensin II–induced hypertension. Using T-cell receptor spectratyping to examine T-cell receptor usage, we demonstrated that CD8+ cells, but not CD4+ cells, in the kidney exhibited altered T-cell receptor transcript lengths in V&bgr;3, 8.1, and 17 families in response to angiotensin II–induced hypertension. Clonality was not observed in other organs. The hypertension caused by angiotensin II in CD4−/− and MHCII−/− mice was similar to that observed in wild-type mice, whereas CD8−/− mice and OT1xRAG-1−/− mice, which have only 1 T-cell receptor, exhibited a blunted hypertensive response to angiotensin II. Adoptive transfer of pan T cells and CD8+ T cells but not CD4+/CD25− cells conferred hypertension to RAG-1−/− mice. In contrast, transfer of CD4+/CD25+ cells to wild-type mice receiving angiotensin II decreased blood pressure. Mice treated with angiotensin II exhibited increased numbers of kidney CD4+ and CD8+ T cells. In response to a sodium/volume challenge, wild-type and CD4−/− mice infused with angiotensin II retained water and sodium, whereas CD8−/− mice did not. CD8−/− mice were also protected against angiotensin-induced endothelial dysfunction and vascular remodeling in the kidney. These data suggest that in the development of hypertension, an oligoclonal population of CD8+ cells accumulates in the kidney and likely contributes to hypertension by contributing to sodium and volume retention and vascular rarefaction.

Lymphocyte adaptor protein LNK deficiency exacerbates hypertension and end-organ inflammation

The lymphocyte adaptor protein LNK (also known as SH2B3) is primarily expressed in hematopoietic and endothelial cells, where it functions as a negative regulator of cytokine signaling and cell proliferation. Single-nucleotide polymorphisms in the gene encoding LNK are associated with autoimmune and cardiovascular disorders; however, it is not known how LNK contributes to hypertension. Here, we determined that loss of LNK exacerbates angiotensin II-induced (Ang II-induced) hypertension and the associated renal and vascular dysfunction. At baseline, kidneys from Lnk-/- mice exhibited greater levels of inflammation, oxidative stress, and glomerular injury compared with WT animals, and these parameters were further exacerbated by Ang II infusion. Aortas from Lnk-/- mice exhibited enhanced inflammation, reduced nitric oxide levels, and impaired endothelial-dependent relaxation. Bone marrow transplantation studies demonstrated that loss of LNK in hematopoietic cells is primarily responsible for the observed renal and vascular inflammation and predisposition to hypertension. Ang II infusion increased IFN-γ-producing CD8+ T cells in the spleen and kidneys of Lnk-/- mice compared with WT mice. Moreover, IFN-γ deficiency resulted in blunted hypertension in response to Ang II infusion. Together, these results suggest that LNK is a potential therapeutic target for hypertension and its associated renal and vascular sequela.

Renal Denervation Prevents Immune Cell Activation and Renal Inflammation in Angiotensin II–Induced Hypertension

RATIONALE Inflammation and adaptive immunity play a crucial role in the development of hypertension. Angiotensin II and probably other hypertensive stimuli activate the central nervous system and promote T-cell activation and end-organ damage in peripheral tissues. OBJECTIVE To determine if renal sympathetic nerves mediate renal inflammation and T-cell activation in hypertension. METHODS AND RESULTS Bilateral renal denervation using phenol application to the renal arteries reduced renal norepinephrine levels and blunted angiotensin II-induced hypertension. Bilateral renal denervation also reduced inflammation, as reflected by decreased accumulation of total leukocytes, T cells, and both CD4+ and CD8+ T cells in the kidney. This was associated with a marked reduction in renal fibrosis, albuminuria, and nephrinuria. Unilateral renal denervation, which partly attenuated blood pressure, only reduced inflammation in the denervated kidney, suggesting that this effect is pressure independent. Angiotensin II also increased immunogenic isoketal-protein adducts in renal dendritic cells (DCs) and increased surface expression of costimulation markers and production of interleukin (IL)-1α, IL-1β, and IL-6 from splenic DCs. Norepinephrine also dose dependently stimulated isoketal formation in cultured DCs. Adoptive transfer of splenic DCs from angiotensin II-treated mice primed T-cell activation and hypertension in recipient mice. Renal denervation prevented these effects of hypertension on DCs. In contrast to these beneficial effects of ablating all renal nerves, renal afferent disruption with capsaicin had no effect on blood pressure or renal inflammation. CONCLUSIONS Renal sympathetic nerves contribute to DC activation, subsequent T-cell infiltration and end-organ damage in the kidney in the development of hypertension.

Renal Transporter Activation During Angiotensin-II Hypertension is Blunted in Interferon-γ−/− and Interleukin-17A−/− Mice

Ample genetic and physiological evidence establishes that renal salt handling is a critical regulator of blood pressure. Studies also establish a role for the immune system, T-cell infiltration, and immune cytokines in hypertension. This study aimed to connect immune cytokines, specifically interferon-&ggr; (IFN-&ggr;) and interleukin-17A (IL-17A), to sodium transporter regulation in the kidney during angiotensin-II (Ang-II) hypertension. C57BL/6J (wild-type) mice responded to Ang-II infusion (490 ng/kg per minute, 2 weeks) with a rise in blood pressure (170 mm Hg) and a significant decrease in the rate of excretion of a saline challenge. In comparison, mice that lacked the ability to produce either IFN-&ggr; (IFN-&ggr;−/−) or IL-17A (IL-17A−/−) exhibited a blunted rise in blood pressure (<150 mm Hg), and both the genotypes maintained baseline diuretic and natriuretic responses to a saline challenge. Along the distal nephron, Ang-II infusion increased abundance of the phosphorylated forms of the Na-K-2Cl cotransporter, Na-Cl cotransporter, and Ste20/SPS-1–related proline-alanine–rich kinase, in both the wild-type and the IL-17A−/− but not in IFN-&ggr;−/− mice; epithelial Na channel abundance increased similarly in all the 3 genotypes. In the proximal nephron, Ang-II infusion significantly decreased abundance of Na/H-exchanger isoform 3 and the motor myosin VI in IL-17A−/− and IFN-&ggr;−/−, but not in wild-type; the Na-phosphate cotransporter decreased in all the 3 genotypes. Our results suggest that during Ang-II hypertension both IFN-&ggr; and IL-17A production interfere with the pressure natriuretic decrease in proximal tubule sodium transport and that IFN-&ggr; production is necessary to activate distal sodium reabsorption.

Inflammation and Mechanical Stretch Promote Aortic Stiffening in Hypertension Through Activation of p38 Mitogen-Activated Protein KinaseNovelty and Significance

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 1&agr;1, 3&agr;1, 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.

Immune activation caused by vascular oxidation promotes fibrosis and hypertension

Vascular oxidative injury accompanies many common conditions associated with hypertension. In the present study, we employed mouse models with excessive vascular production of ROS (tg(sm/p22phox) mice, which overexpress the NADPH oxidase subunit p22(phox) in smooth muscle, and mice with vascular-specific deletion of extracellular SOD) and have shown that these animals develop vascular collagen deposition, aortic stiffening, renal dysfunction, and hypertension with age. T cells from tg(sm/p22phox) mice produced high levels of IL-17A and IFN-γ. Crossing tg(sm/p22phox) mice with lymphocyte-deficient Rag1(-/-) mice eliminated vascular inflammation, aortic stiffening, renal dysfunction, and hypertension; however, adoptive transfer of T cells restored these processes. Isoketal-protein adducts, which are immunogenic, were increased in aortas, DCs, and macrophages of tg(sm/p22phox) mice. Autologous pulsing with tg(sm/p22phox) aortic homogenates promoted DCs of tg(sm/p22phox) mice to stimulate T cell proliferation and production of IFN-γ, IL-17A, and TNF-α. Treatment with the superoxide scavenger tempol or the isoketal scavenger 2-hydroxybenzylamine (2-HOBA) normalized blood pressure; prevented vascular inflammation, aortic stiffening, and hypertension; and prevented DC and T cell activation. Moreover, in human aortas, the aortic content of isoketal adducts correlated with fibrosis and inflammation severity. Together, these results define a pathway linking vascular oxidant stress to immune activation and aortic stiffening and provide insight into the systemic inflammation encountered in common vascular diseases.

Distinct Actions of Endothelin A-Selective Versus Combined Endothelin A/B Receptor Antagonists in Early Diabetic Kidney Disease

Selective endothelin A (ETA) and combined ETA and ETB receptor antagonists are being investigated for use in treating diabetic nephropathy. However, the receptor-specific mechanisms responsible for producing the potential benefits have not been discerned. Thus, we determined the actions of ETA and ETB receptors on measures of glomerular function and renal inflammation in the early stages of diabetic renal injury in rats through the use of selective and combined antagonists. Six weeks after streptozotocin (STZ)-induced hyperglycemia, rats were given 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627) (5 mg/kg/day), a selective ETA antagonist; (2R,3R,4S)-4-(benzo[d][1,3]dioxol-5-yl)-2-(3-fluoro-4-methoxyphenyl)-1-(2-(N-propylpentylsulfonamido)ethyl)pyrrolidine-3-carboxylic acid hydrochloride (A-182086) (10 mg/kg/day), a combined ETA/B antagonist; or vehicle for 1 week. Sham controls received STZ vehicle (saline). Hyperglycemia led to significant proteinuria, increased glomerular permeability to albumin (Palb), nephrinuria, and an increase in total matrix metalloprotease (MMP) and transforming growth factor-β1 (TGF-β1) activities in glomeruli. Plasma and glomerular soluble intercellular adhesion molecule-1 (sICAM-1) and monocyte chemoattractant protein-1 (MCP-1) were elevated after 7 weeks of hyperglycemia. Daily administration of both ABT-627 and A-182086 for 1 week significantly attenuated proteinuria, the increase in Palb, nephrinuria, and total MMP and TGF-β1 activity. However, glomerular sICAM-1 and MCP-1 expression was attenuated with ABT-627, but not A-182086, treatment. In summary, both selective ETA and combined ETA/B antagonists reduced proteinuria and glomerular permeability and restored glomerular filtration barrier component integrity, but only ETA-selective blockade had anti-inflammatory and antifibrotic effects. We conclude that selective ETA antagonists are more likely to be preferred for the treatment of diabetic kidney disease.

Integrative network analysis reveals molecular mechanisms of blood pressure regulation.

Genome‐wide association studies (GWAS) have identified numerous loci associated with blood pressure (BP). The molecular mechanisms underlying BP regulation, however, remain unclear. We investigated BP‐associated molecular mechanisms by integrating BP GWAS with whole blood mRNA expression profiles in 3,679 individuals, using network approaches. BP transcriptomic signatures at the single‐gene and the coexpression network module levels were identified. Four coexpression modules were identified as potentially causal based on genetic inference because expression‐related SNPs for their corresponding genes demonstrated enrichment for BP GWAS signals. Genes from the four modules were further projected onto predefined molecular interaction networks, revealing key drivers. Gene subnetworks entailing molecular interactions between key drivers and BP‐related genes were uncovered. As proof‐of‐concept, we validated SH2B3, one of the top key drivers, using Sh2b3−/− mice. We found that a significant number of genes predicted to be regulated by SH2B3 in gene networks are perturbed in Sh2b3−/− mice, which demonstrate an exaggerated pressor response to angiotensin II infusion. Our findings may help to identify novel targets for the prevention or treatment of hypertension.