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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.

Metabolites produced by the microbiome such as short chain fatty acids (SCFAs) and tryptophan metabolites have been shown to impact the pathogenesis of hypertension. The microenvironment of the host gastrointestinal (GI) tract acts as the site-of-action for many host-microbiome interactions, although this space is not readily accessible. Fecal or serum samples are commonly used as a proxy, relying on the assumption that the obtained information should in some respect represent what is seen at the host epithelial interface. We surmised that it would be feasible to overcome the limitations of such indirect analysis by isolating interstitial fluid (IF) from the gut mucosa. We have established two methodologies to isolate IF from small segments of along GI tract, a centrifugation-based and elution-based approach in rats and mice. For rats and mice, 51 Cr-EDTA tracer experiments were used to demonstrate that fluid obtained was from an extracellular origin and can be reliably considered IF. Using GC-MS, we could identify several SCFAs (acetate, butyrate, propionate, valerate) within eluted IF samples. We were able to empirically measure the enrichment of these metabolites in eluted IF from the colon of rats compared to the duodenum (for propionate; mean difference=59.57 μM, p-value < 0.0001) or the serum (for propionate; mean difference= 60.25 μM, p-value < 0.0001). LC-MS based shotgun proteomics revealed that proteins annotated to the extracellular phase were site-specifically identifiable in IF and were differentially expressed when compared to matched serum samples. Furthermore, we could demonstrate that the use of tissue for IF isolation does not impede the use of other methods such as immunophenotyping or histology. Absolute CD45+ cells from the lamina propria measured using flow cytometry were not influenced by IF methods (p-value = 0.8344) but were unsurprisingly influenced by the gut segment which was analyzed (p-value = 0.0010). The ability to collect IF and directly measure metabolites at the site-of-action overcomes the limitations of indirect analysis of fecal samples or serum from the systemic circulation, and thus offers direct insight into this hitherto underexplored compartment.

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.