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Background: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A ( PDE3A ); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking. Methods: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways. Results: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function. Conclusions: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.

AimsHypertension (HTN) can lead to heart and kidney damage. The gut microbiota has been linked to HTN, although it is difficult to estimate its significance due to the variety of other features known to influence HTN. In the present study, we used germ-free (GF) and colonized (COL) littermate mice to quantify the impact of microbial colonization on organ damage in HTN.Methods and resultsFour-week-old male GF C57BL/6J littermates were randomized to remain GF or receive microbial colonization. HTN was induced by subcutaneous infusion with angiotensin (Ang) II (1.44mg/kg/d) and 1% NaCl in the drinking water; sham-treated mice served as control. Renal damage was exacerbated in GF mice, whereas cardiac damage was more comparable between COL and GF, suggesting that the kidney is more sensitive to microbial influence. Multivariate analysis revealed a larger effect of HTN in GF mice. Serum metabolomics demonstrated that the colonization status influences circulating metabolites relevant to HTN. Importantly, GF mice were deficient in anti-inflammatory fecal short-chain fatty acids (SCFA). Flow cytometry showed that the microbiome has an impact on the induction of anti-hypertensive myeloid-derived suppressor cells and pro-inflammatory Th17 cells in HTN. In vitro inducibility of Th17 cells was significantly higher for cells isolated from GF than conventionally raised mice.ConclusionsMicrobial colonization status of mice had potent effects on their phenotypic response to a hypertensive stimulus, and the kidney is a highly microbiota-susceptible target organ in HTN. The magnitude of the pathogenic response in GF mice underscores the role of the microbiome in mediating inflammation in HTN.Translation PerspectiveTo assess the potential of microbiota-targeted interventions to prevent organ damage in hypertension, an accurate quantification of microbial influence is necessary. We provide evidence that the development of hypertensive organ damage is dependent on colonization status and suggest that a healthy microbiota provides anti-hypertensive immune and metabolic signals to the host. In the absence of normal symbiotic host-microbiome interactions, hypertensive damage to the kidney in particular is exacerbated. We suggest that hypertensive patients experiencing perturbations to the microbiota, which are common in CVD, may be at a greater risk for target-organ damage than those with a healthy microbiome.

Introduction: The gut microbiota is suspected to play a role in hypertension and hypertensive end organ damage. In the present study, we used germ-free mice to demonstrate that microbial colonization modulates the response to a hypertensive stimulus. Methods: Four-week-old male germ-free C57BL6/J littermates were randomized to remain germ-free (GF) or to receive microbiota transfer from SPF donor mice to achieve full colonization status (COL). At 12 weeks, Angiotensin (Ang) II was infused s.c. for 14 days (1.44mg/kg/d, osmotic minipumps) and 1% NaCl added to the drinking water; sham-treated mice served as control. After 14 days of AngII we assessed inflammation and organ damage. Results: Fecal bacterial load in COL mice was similar to SPF donor mice (qPCR). Shotgun metagenomic sequencing of fecal samples revealed hypertension-induced alterations in microbiome composition confirming previous reports. Serum metabolome analysis ( Biocrates MxP Quant 500) confirmed the absence of microbiota-dependent metabolites in GF. Interestingly, microbiota-dependent metabolites relevant for cardiovascular risk (TMAO, indoxyl sulfate) were elevated in hypertensive COL mice compared to sham-treated. Hypertensive kidney damage was aggravated in GF mice. However, marker genes for tubular damage ( Lcn2 ), inflammation ( Ccl2 ), and fibrosis ( Col1a3 ) showed a stronger increase in GF mice (fold changes [fc] COL vs. GF: 7.5 vs 11.0, 1.2 vs 3.3, 1.3 vs 2.2, respectively). Albuminuria (fc 2 vs 25) and histology for kidney fibrosis (fc 1.1 vs 1.4) confirmed the aggravated kidney damage in GF mice. Similarly, we observed an aggravated cardiac damage in GF mice. Flow cytometry of splenic lymphocytes showed that the adaptive immune response to AngII + 1% NaCl, as evidenced by Th17 (fc 1.4 vs 2) and CD8+ central memory cells, was intensified in GF mice. In vitro , naïve T cells isolated from GF mice more readily polarized into Th17 (26 ± 5%) compared to T cells from SPF mice (19 ± 1%). Conclusion: The bacterial colonization status has potent effects on the phenotypic response to a hypertensive stimulus, evident to varying degrees in hearts and kidneys. The inflammatory response and the end organ damage in GF compared to COL mice demonstrates the importance of the gut microbiota in hypertension.