Jessica R. C. Priestley

Mutation of Plekha7 attenuates salt-sensitive hypertension in the rat

Significance Zinc-finger nuclease (ZFN)-mediated mutagenesis has now enabled researchers to manipulate specific genes to test their function in animal models other than mice. Applying ZFNs to rats, we can now test the role of specific human genome-wide association studies (GWAS)-nominated genes for hypertension in a well-characterized hypertensive rat model, the Dahl salt-sensitive rat. This study provides the first functional evidence that the GWAS-nominated gene Plekha7 plays an essential role in blood pressure regulation and cardiovascular function by modulating vascular function. Our results indicate that Plekha7 plays a role in the regulation of intracellular calcium, nitric oxide bioavailability, and the response of the vasculature to increased flow. PLEKHA7 (pleckstrin homology domain containing family A member 7) has been found in multiple studies as a candidate gene for human hypertension, yet functional data supporting this association are lacking. We investigated the contribution of this gene to the pathogenesis of salt-sensitive hypertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger nuclease technology. After four weeks on an 8% NaCl diet, homozygous mutant rats had lower mean arterial (149 ± 9 mmHg vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± 8 mmHg; P < 0.05) blood pressure compared with WT littermates. Albumin and protein excretion rates were also significantly lower in mutant rats, demonstrating a renoprotective effect of the mutation. Total peripheral resistance and perivascular fibrosis in the heart and kidney were significantly reduced in Plekha7 mutant animals, suggesting a potential role of the vasculature in the attenuation of hypertension. Indeed, both flow-mediated dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT. These vascular improvements were correlated with changes in intracellular calcium handling, resulting in increased nitric oxide bioavailability in mutant vessels. Collectively, these data provide the first functional evidence that Plekha7 may contribute to blood pressure regulation and cardiovascular function through its effects on the vasculature.

Mutation of SH2B3 (LNK), a Genome-Wide Association Study Candidate for Hypertension, Attenuates Dahl Salt-Sensitive Hypertension via Inflammatory Modulation

Human genome-wide association studies have linked SH2B adaptor protein 3 (SH2B3, LNK) to hypertension and renal disease, although little experimental investigation has been performed to verify a role for SH2B3 in these pathologies. SH2B3, a member of the SH2B adaptor protein family, is an intracellular adaptor protein that functions as a negative regulator in many signaling pathways, including inflammatory signaling processes. To explore a mechanistic link between SH2B3 and hypertension, we targeted the SH2B3 gene for mutation on the Dahl salt-sensitive (SS) rat genetic background with zinc-finger nucleases. The resulting mutation was a 6-bp, in-frame deletion within a highly conserved region of the Src homology 2 (SH2) domain of SH2B3. This mutation significantly attenuated Dahl SS hypertension and renal disease. Also, infiltration of leukocytes into the kidneys, a key mediator of Dahl SS pathology, was significantly blunted in the Sh2b3em1Mcwi mutant rats. To determine whether this was because of differences in immune signaling, bone marrow transplant studies were performed in which Dahl SS and Sh2b3em1Mcwi mutants underwent total body irradiation and were then transplanted with Dahl SS or Sh2b3em1Mcwi mutant bone marrow. Rats that received Sh2b3em1Mcwi mutant bone marrow had a significant reduction in mean arterial pressure and kidney injury when placed on a high salt diet (4% NaCl). These data further support a role for the immune system as a modulator of disease severity in the pathogenesis of hypertension and provide insight into inflammatory mechanisms at play in human hypertension and renal disease.

The NRF2 knockout rat: a new animal model to study endothelial dysfunction, oxidant stress, and microvascular rarefaction.

Nuclear factor (erythroid-derived 2)-like-2 (NRF2) is a master antioxidant and cell protective transcription factor that upregulates antioxidant defenses. In this study we developed a strain of Nrf2 null mutant rats to evaluate the role of reduced NRF2-regulated antioxidant defenses in contributing to endothelial dysfunction and impaired angiogenic responses during salt-induced ANG II suppression. Nrf2(-/-) mutant rats were developed using transcription activator-like effector nuclease technology in the Sprague-Dawley genetic background, and exhibited a 41-bp deletion that included the start codon for Nrf2 and an absence of immunohistochemically detectable NRF2 protein. Expression of mRNA for the NRF2-regulated indicator enzymes heme oxygenase-1, catalase, superoxide dismutase 1, superoxide dismutase 2, and glutathione reductase was significantly lower in livers of Nrf2(-/-) mutant rats fed high salt (HS; 4% NaCl) for 2 wk compared with wild-type controls. Endothelium-dependent dilation to acetylcholine was similar in isolated middle cerebral arteries (MCA) of Nrf2(-/-) mutant rats and wild-type littermates fed low-salt (0.4% NaCl) diet, and was eliminated by short-term (3 days) HS diet in both strains. Low-dose ANG II infusion (100 ng/kg sc) reversed salt-induced endothelial dysfunction in MCA and prevented microvessel rarefaction in wild-type rats fed HS diet, but not in Nrf2(-/-) mutant rats. The results of this study indicate that suppression of NRF2 antioxidant defenses plays an essential role in the development of salt-induced oxidant stress, endothelial dysfunction, and microvessel rarefaction in normotensive rats and emphasize the potential therapeutic benefits of directly upregulating NRF2-mediated antioxidant defenses to ameliorate vascular oxidant stress in humans.

Reduced angiotensin II levels cause generalized vascular dysfunction via oxidant stress in hamster cheek pouch arterioles

OBJECTIVES We investigated the effect of suppressing plasma angiotensin II (ANG II) levels on arteriolar relaxation in the hamster cheek pouch. METHODS Arteriolar diameters were measured via television microscopy during short-term (3-6days) high salt (HS; 4% NaCl) diet and angiotensin converting enzyme (ACE) inhibition with captopril (100mg/kg/day). RESULTS ACE inhibition and/or HS diet eliminated endothelium-dependent arteriolar dilation to acetylcholine, endothelium-independent dilation to the NO donor sodium nitroprusside, the prostacyclin analogs carbacyclin and iloprost, and the KATP channel opener cromakalim; and eliminated arteriolar constriction during KATP channel blockade with glibenclamide. Scavenging of superoxide radicals and low dose ANG II infusion (25ng/kg/min, subcutaneous) reduced oxidant stress and restored arteriolar dilation in arterioles of HS-fed hamsters. Vasoconstriction to topically-applied ANG II was unaffected by HS diet while arteriolar responses to elevation of superfusion solution PO2 were unaffected (5% O2, 10% O2) or reduced (21% O2) by HS diet. CONCLUSIONS These findings indicate that sustained exposure to low levels of circulating ANG II leads to widespread dysfunction in endothelium-dependent and independent vascular relaxation mechanisms in cheek pouch arterioles by increasing vascular oxidant stress, but does not potentiate O2- or ANG II-induced constriction of arterioles in the distal microcirculation of normotensive hamsters.

Mutation of SH2B3 (LNK), a GWAS candidate for hypertension, attenuates Dahl SS hypertension via inflammatory modulation

Human genome-wide association studies have linked SH2B adaptor protein 3 (SH2B3, LNK) to hypertension and renal disease, although little experimental investigation has been performed to verify a role for SH2B3 in these pathologies. SH2B3, a member of the SH2B adaptor protein family, is an intracellular adaptor protein that functions as a negative regulator in many signaling pathways, including inflammatory signaling processes. To explore a mechanistic link between SH2B3 and hypertension, we targeted the SH2B3 gene for mutation on the Dahl salt-sensitive (SS) rat genetic background with zinc-finger nucleases. The resulting mutation was a 6-bp, in-frame deletion within a highly conserved region of the Src homology 2 (SH2) domain of SH2B3. This mutation significantly attenuated Dahl SS hypertension and renal disease. Also, infiltration of leukocytes into the kidneys, a key mediator of Dahl SS pathology, was significantly blunted in the Sh2b3em1Mcwi mutant rats. To determine whether this was because of differences in immune signaling, bone marrow transplant studies were performed in which Dahl SS and Sh2b3em1Mcwi mutants underwent total body irradiation and were then transplanted with Dahl SS or Sh2b3em1Mcwi mutant bone marrow. Rats that received Sh2b3em1Mcwi mutant bone marrow had a significant reduction in mean arterial pressure and kidney injury when placed on a high salt diet (4% NaCl). These data further support a role for the immune system as a modulator of disease severity in the pathogenesis of hypertension and provide insight into inflammatory mechanisms at play in human hypertension and renal disease.

Vascular dysfunction precedes hypertension associated with a blood pressure locus on rat chromosome 12

We previously isolated a 6.1-Mb region of SS/Mcwi (Dahl salt-sensitive) rat chromosome 12 (13.4-19.5 Mb) that significantly elevated blood pressure (BP) (Δ+34 mmHg, P < 0.001) compared with the SS-12(BN) consomic control. In the present study, we examined the role of vascular dysfunction and remodeling in hypertension risk associated with the 6.1-Mb (13.4-19.5 Mb) locus on rat chromosome 12 by reducing dietary salt, which lowered BP levels so that there were no substantial differences in BP between strains. Consequently, any observed differences in the vasculature were considered BP-independent. We also reduced the candidate region from 6.1 Mb with 133 genes to 2 Mb with 23 genes by congenic mapping. Both the 2 Mb and 6.1 Mb congenic intervals were associated with hypercontractility and decreased elasticity of resistance vasculature prior to elevations of BP, suggesting that the vascular remodeling and dysfunction likely contribute to the pathogenesis of hypertension in these congenic models. Of the 23 genes within the narrowed congenic interval, 12 were differentially expressed between the resistance vasculature of the 2 Mb congenic and SS-12(BN) consomic strains. Among these, Grifin was consistently upregulated 2.7 ± 0.6-fold (P < 0.05) and 2.0 ± 0.3-fold (P < 0.01), and Chst12 was consistently downregulated -2.8 ± 0.3-fold (P < 0.01) and -4.4 ± 0.4-fold (P < 0.00001) in the 2 Mb congenic compared with SS-12(BN) consomic under normotensive and hypertensive conditions, respectively. A syntenic region on human chromosome 7 has also been associated with BP regulation, suggesting that identification of the genetic mechanism(s) underlying cardiovascular phenotypes in this congenic strain will likely be translated to a better understanding of human hypertension.

Mutation of SH2B3 (LNK), a Genome-Wide Association Study Candidate for Hypertension, Attenuates Dahl Salt-Sensitive Hypertension via Inflammatory ModulationNovelty and Significance

Human genome-wide association studies have linked SH2B adaptor protein 3 (SH2B3, LNK) to hypertension and renal disease, although little experimental investigation has been performed to verify a role for SH2B3 in these pathologies. SH2B3, a member of the SH2B adaptor protein family, is an intracellular adaptor protein that functions as a negative regulator in many signaling pathways, including inflammatory signaling processes. To explore a mechanistic link between SH2B3 and hypertension, we targeted the SH2B3 gene for mutation on the Dahl salt-sensitive (SS) rat genetic background with zinc-finger nucleases. The resulting mutation was a 6-bp, in-frame deletion within a highly conserved region of the Src homology 2 (SH2) domain of SH2B3. This mutation significantly attenuated Dahl SS hypertension and renal disease. Also, infiltration of leukocytes into the kidneys, a key mediator of Dahl SS pathology, was significantly blunted in the Sh2b3em1Mcwi mutant rats. To determine whether this was because of differences in immune signaling, bone marrow transplant studies were performed in which Dahl SS and Sh2b3em1Mcwi mutants underwent total body irradiation and were then transplanted with Dahl SS or Sh2b3em1Mcwi mutant bone marrow. Rats that received Sh2b3em1Mcwi mutant bone marrow had a significant reduction in mean arterial pressure and kidney injury when placed on a high salt diet (4% NaCl). These data further support a role for the immune system as a modulator of disease severity in the pathogenesis of hypertension and provide insight into inflammatory mechanisms at play in human hypertension and renal disease.