Abstract 4347857: Decreased nitrate reduction capacity of oral microbiota is associated with increased severity of pulmonary hypertension

Background: Pulmonary hypertension (PH) is a fatal condition characterized by elevated pressure in pulmonary artery which could be partly caused by dysfunctional nitric oxide (NO) signaling. Previous studies identified that nitrate is actively concentrated in saliva and could be reduced to nitrite by bacterial nitrate reductase enzymes, which are rarely found in human genomes. Oral nitrate reduction (NR) capacity was previously reported to be predictive of blood pressure in systemic circulation; however, the role of the oral microbiome in oral NR and NO signaling in PH remains unclear. Hypothesis: We hypothesize that loss of nitrate reductase-expressing bacteria in oral microbiota leads to decreased NR capacity and is associated with worse PH phenotype. Method: We prospectively enrolled patients who underwent RHC at University of Pittsburgh Medical Center, following the ERS 2022 PH hemodynamic classification. Oral wash [OW] samples were collected&underwent 16S rRNA sequencing then analyzed with Qiime2 (v2024.10; SILVA). We predicted bacterial NR capacity in silico by Picrust2 (v2.6). Differential abundance analyses were done with ANCOMBC2. Plasma nitrate&nitrite levels were quantified. In a subset, NR capacity was verified by measuring ex vivo nitrite formation rate with NO analyzer (Sievers) after adding KNO 3 substrate to OW. Results: We enrolled n=184 patients who underwent RHC in 2014-2020. Of these, 144 (78%) had mPAP over 20mmHg (PH) with mPAP of 32.4 [10.2] mmHg (mean [SD]) and PVR of 3.6 [1.9] Wood Units. Between PH vs non-PH patients, alpha diversity, beta diversity and microbial load (16S qPCR) were similar in the oral microbiome (all p>0.05). Veillonella, Rothia, Neisseria etc are major NR capacity contributors, but their abundances did not differ between PH vs non-PH. NR index, defined as the ratio of sum abundance of NR contributors over non-contributors, was significantly associated with the predicted abundance of bacterial nitrate reductase genes, and also verified with the NR enzyme activity (p=0.016) measured in vitro . Increased NR index is associated with lower mPAP (R=-0.21, p=0.0129, adjusted for diastolic systematic blood pressure [DBP] and smoking history) and lower PVR (R= -0.19, adjusted p=0.019) among PH patients. Conclusions: We confirmed that predicted nitrate reduction capacity by the oral microbiome reflects functional nitrate reduction in vitro and is associated with PH severity. The oral microbiome represents a modifiable target in PH.