Preparation of a New Nanobody and Its Usage in Treatment of Spontaneous Hypertension

In order to improve the controllability of spontaneous hypertension, it was necessary to study a new nanobody that can inhibit calcium flow and reduce hypertension by considering the specificity and long half-life of antibody. Firstly, the antibody epitope screening technology was adopted to select a short peptide (CN8) in the E3 region of the first domain of Cav1.2 α1c subunit, the full-length complementary deoxyribonucleic acid (DNA) (cDNA) clone of which was undertaken as a template. The gene fragment in extracellular region of CN8 was amplified by polymerase chain reaction (PCR), and the pET30a-CN8 recombinant plasmid was constructed. Ribose nucleic acids (RNAs) in peripheral lymphocytes of 5 camels were extracted, and the reverse transcribed cDNA was taken as a template to realize the amplification of VHH gene fragment. After the double enzyme digestion, the pMECS vector was connected and TG1 was transformed to form a natural VHH antibody library. The phage display library was screened by CN8 protein to obtain the new nanobody (VHH-CN8). Western blotting (WB) and immunofluorescence (IF) were used to analyze the specific binding of antibody, and the calcium flux test was adopted to test the effect of antibody on calcium channels. A rat model of spontaneous hypertension and a mice model of spontaneous hypertension induced by angiotensin II (Ang II) were constructed to verify the antihypertensive effect of the prepared nanobody materials. In vitro, the VHH-CN8 antibody could realize specific binding to the Cav1.2 channel, inhibit the transfected human embryonic kidney (HEK293) cells, and prevent the increase in intracellular calcium concentration of arterial smooth muscle by adjusting the concentration and potential; in vivo, the VHH-CN8 antibody could reduce the blood pressure level of spontaneously hypertensive rats (SHRs) and mice in Ang II model, which was 196±3/186±4 mmHg (P < 0.05) and 187±5/158±3 mmHg (P < 0.01), respectively. Thus, it proved that the prepared nanobody could reduce the blood pressure level of spontaneous hypertension.

Abstract 22: Formyl Peptide Receptor-1 Activation Is Crucial For The Cause Of Spontaneous Hypertension In Dahl Salt Sensitive Rats

Mitochondria evolved from bacteria and use N-formylated peptides (NFPs) to synthetize protein. Bacterial and mitochondrial NFPs activate formyl peptide receptor 1 (FPR-1) and lead to vascular injury. We previously observed that Dahl Salt Sensitive rats (S) fed a low-salt (LS, 0.3% NaCl) diet presented spontaneous hypertension, vascular dysfunction, and overexpression of FPR-1 in arteries when compared to Dahl Salt Resistant (R) rats. High salt (HS, 2% NaCl) diet worsened these phenotypes in S rats. Interestingly, HS diet induced leaky gut and amoxicillin (AMO) treatment decreased BP in S-HS. Due to the dual sources of NFPs (microbiota and host mitochondria), we hypothesized that cell death-derived mitochondria and/or leaky gut-derived bacterial NFPs lead to FPR-1 activation, vascular injury and elevated BP in S rats independent of HS diet. For this, we used flow cytometry to measure cell necrosis and early and late apoptosis in kidney, bone marrow-derived macrophages and mesenteric resistance arteries (MRA) from male S and R rats (8-week old) on a LS diet. Zonulin, a biomarker for leaky gut, was measured in plasma. In another group, rats were treated with FPR-1 antagonist [Cyclosporin H (CsH), 0.3 mg/kg/day, osmotic mini-pump, 14 days], vehicle (VEH) or received water with AMO (5 mg/kg/day) for 21 days to deplete bacteria. BP was measured by telemetry and vascular function and structure were assessed in MRA. S rats presented increased kidney cell necrosis (R: 3.8±0.3 vs. S: 5.3±0.5* %). CsH decreased spontaneous elevation of BP [Diastolic: R+VEH: 77±2.7 vs. R+CsH: 81±1.2 vs. S+VEH: 126±3.0* vs. S+CsH:115±2.7 # ] and vascular hypercontractility [KCl (120mM): R+VEH: 9.4±1 vs. R+CsH: 10.2±0.4; S+VEH: 15.5±0.9* vs. S+CsH:11.7±0.8 # mN; Phenylephrine (10μM): R+VEH: 9.3±1 vs. R+CsH: 9.7±1; S+VEH: 14.5±1*vs. S+CsH: 11.4±0.6 # mN) in S-LS rats. AMO did not change vascular contraction or BP. Leaky gut was not observed in Dahl S-LS diet. In conclusion, FPR-1 can serve as a causative agent for the spontaneous elevation of BP and kidney-derived mitochondria, but not gut-derived microbiota, are the main source for NFPs.

P2256Aging and inhibition of Nox4 and Nox1 exacerbate perivascular inflammation in spontaneous hypertension

Introduction Hypertension is associated with enhanced NADPH oxidase activation and increased accumulation of immune cells leading to perivascular inflammation. However, while aging and oxidative stress are a major factor for the development of hypertension, their effect on perivascular inflammation remains unclear. Purpose We aimed to establish the interaction between aging, oxidative stress and perivascular inflammation. We hypothesize that the modulation of oxidative stress, by NADPH oxidase inhibition, could affect perivascular inflammation during the aging. Methods Using flow cytometry, we studied leukocyte infiltration in the perivascular adipose tissue (PVAT) in 1-, 3-, 6- and 12-month-old SHR (Spontaneously Hypertensive Rats) and normotensive WKY (Wistar-Kyoto) rats (n=5–12). Additionally, 1-month-old rats were treated with GKT137831 or ML171 (60mg/kg; NOX1/4 and NOX1 inhibitor, respectively) for 4 weeks. Blood pressure (PB) was measured by tail cuff. Statistical analysis was performed using two-way ANOVA or t-test. Data are presented as means±SEM. Results Aging in SHRs was associated with an increase of BP (139±4 vs. 180±4 vs. 203±3 vs. 209±3, mmHg) and an elevation of PVAT leukocytes (2090±164 vs. 2255±359 vs. 2502±496 vs. 3255±408, cell/mg) in 1-, 3-, 6- and 12-month-old rats, respectively. These effects were not seen in WKY. These were associate with similar changes of NK cells (p int <0.001) and macrophages (p int <0.001) in the SHR PVAT. Within T cell compartment, TH17 cells in PVAT were only elevated with age in SHR (1.04±0.08% vs. 1.89±0.2%, 1.08±0.1% vs. 2.03±0.2%, 1.71% vs. 4.37±0.5%, WKY vs. SHR, 3-,6- and 12-months, respectively, p int<0.001). While an age-related increase of Nox4 mRNA in the vessels was observed in both groups, this increase was more dynamic in SHRs, (p int <0.05). Furthermore, 5-, 6- and 9-fold induction of Nox1 mRNA was observed in the vessels of 3-, 6- and 12-months-old SHRs, respectively (p<0.01). Interestingly, GKT137831 increased BP in both WKY and SHR rats (p<0.01, 2-way ANOVA). This was accompanied by the elevation of leukocytes (988±180 vs 147±188 cell/mg, 1487±945 vs 1878±164 cell/mg) and macrophages (107±14 vs. 153±14 cell/mg, 228±26 vs. 298±42 cell/mg) in PVAT of both WKYs and SHRs treated with GKT137831. Moreover, GKT137831 treatment significantly increased (p int<0.01) percentage of TH17 in PVAT of both normotensive (1.4±0.2% vs. 2.5±0.2%) and hypertensive (2.3±0.1% vs. 2.9±0.1%) animals. On the contrary, ML171 treatment, inhibiting only Nox1, in turn protected against an increase of PVAT leukocytes especially among macrophages, TH17 cells (p<0.01). Conclusion Aging and spontaneous hypertension is associated with a trajectory of BP elevation and perivascular inflammation. This state is hastened after NOX1/4 inhibition, while ML171 treatment protects against perivascular inflammation. Acknowledgement/Funding National Science Centre 2013/11/N/NZ4/00310