Targeting cytokine-like protein FAM3D lowers blood pressure in hypertension

Abstract

Current antihypertensive options still incompletely control blood pressure, suggesting the existence of uncovered pathogenic mechanisms. Here, whether cytokine-like protein family with sequence similarity 3, member D (FAM3D) is involved in hypertension etiology is evaluated. A case-control study exhibits that FAM3D is elevated in patients with hypertension, with a positive association with odds of hypertension. FAM3D deficiency significantly ameliorates angiotensin II (AngII)-induced hypertension in mice. Mechanistically, FAM3D directly causes endothelial nitric oxide synthase (eNOS) uncoupling and impairs endothelium-dependent vasorelaxation, whereas 2,4-diamino-6-hydroxypyrimidine to induce eNOS uncoupling abolishes the protective effect of FAM3D deficiency against AngII-induced hypertension. Furthermore, antagonism of formyl peptide receptor 1 (FPR1) and FPR2 or the suppression of oxidative stress blunts FAM3D-induced eNOS uncoupling. Translationally, targeting endothelial FAM3D by adeno-associated virus or intraperitoneal injection of FAM3D-neutralizing antibodies markedly ameliorates AngII- or deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Conclusively, FAM3D causes eNOS uncoupling through FPR1- and FPR2-mediated oxidative stress, thereby exacerbating the development of hypertension. FAM3D may be a potential therapeutic target for hypertension.

Keywords: FAM3D; antihypertensive treatment; endothelial dysfunction; hypertension.

Graphical abstract

Figure 1

The elevation of FAM3D correlates with the pathogenesis of hypertension (A) ELISA measurement of plasma FAM3D from individuals in the case-control study. N = 80 pairs. Data are represented as mean ± SEM. Mann-Whitney test. (B) Spearman correlation analysis of plasma FAM3D levels and SBP in patients with hypertension. N = 80. (C) ELISA measurement of plasma FAM3D levels in C57BL/6 mice treated with saline or AngII for 7 days. n = 5–6. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (D and E) Immunofluorescent staining of FAM3D in mesenteric arteries (E) and thoracic aortas (F) from C57BL/6 mice after saline or AngII infusion for 7 days. Scale bar, 40 μm. (F) Immunofluorescent en face staining of FAM3D in the endothelial layer of thoracic aortas of C57BL/6 mice after saline or AngII infusion for 1 day or 7 days. Scale bar, 25 μm. n = 6. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (G) Immunofluorescent en face staining of FAM3D in the endothelial layer of thoracic aortas of sham-treated or DOCA-salt-induced mice. Scale bar, 25 μm. n = 6. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (H) Immunofluorescent en face staining of FAM3D in the endothelial layer of thoracic aortas of Wistar-Kyoto (WKY) or SHRs. Scale bar, 25 μm. n = 6. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05.

Figure 2

FAM3D deficiency ameliorates AngII-induced hypertension and related vascular pathologies (A) SBP of WT and FAM3D^−/− mice was measured by the tail-cuff method during AngII infusion for 14 days. n = 6–8. Data are represented as mean ± SEM. Repeated-measures analysis using a mixed-effects model, ∗p < 0.05. (B–D) The SBP (B), DBP (C), and MAP (D) of WT and FAM3D^−/− mice were measured by radiotelemetry during AngII infusion for 14 days. n = 5–6. Data are represented as mean ± SEM. Repeated-measures analysis using a mixed-effects model, ∗p < 0.05. (E and F) 24-h recordings of SBP (E) and DBP (F) in WT and FAM3D^−/− mice was measured by radiotelemetry following 7 days of AngII infusion. n = 3–5. Data are represented as mean ± SEM. Repeated-measures analysis using a mixed-effects model, ∗p < 0.05. (G) Pulse wave velocity (PWV) in aortas from WT and FAM3D^−/− mice treated with saline or AngII for 14 days. n = 5–6. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (H) Representative Masson staining and quantification of adventitial collagen deposition in thoracic aortas from WT and FAM3D^−/− mice treated with saline or AngII for 14 days. Scale bar, 100 μm. n = 5–6. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (I) Flow cytometric analysis of CD45⁺ cells in aortas from WT and FAM3D^−/− mice treated with saline or AngII for 14 days. n = 4–6. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05.

Figure 3

FAM3D impairs endothelium-dependent vasorelaxation to further enhance vasocontraction (A and B) Concentration-response curves of endothelium-dependent contraction (A) and endothelium-independent contraction (B) of mesenteric resistance arteries from WT and FAM3D^−/− mice. n = 6. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test. (C and D) Concentration-response curves of endothelium-dependent contraction (C) and endothelium-independent contraction (D) of mesenteric resistance arteries from WT and FAM3D^−/− mice infused with AngII for 14 days. n = 6. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test. ∗p < 0.05. (E and F) Concentration-response curves of endothelium-dependent relaxation (E) and endothelium-independent relaxation (F) of mesenteric resistance arteries from WT and FAM3D^−/− mice. n = 5–6. Data are represented as mean ± SEM. Two-way ANOVA by Tukey’s multiple comparisons test. (G and H) Concentration-response curves of endothelium-dependent relaxation (G) and endothelium-independent relaxation (H) of mesenteric arteries from WT and FAM3D^−/− mice infused with AngII for 14 days. n = 6. Data are represented as mean ± SEM. Two-way ANOVA by Tukey’s multiple comparisons test. ∗p < 0.05.

Figure 4

FAM3D deficiency inhibits AngII-induced eNOS uncoupling in ECs of aortas in mice (A) Representative DAF-FM DA en face staining and quantification of NO levels in ECs from the thoracic aortas of WT and FAM3D^−/− mice after saline or AngII infusion for 14 days. n = 5–6. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (B) Representative DHE en face staining and quantification of O₂⁻ in ECs from thoracic aortas in WT and FAM3D^−/− mice infused with saline or AngII for 14 days. L-NAME (200 μmol/L) was ex vivo used to treat dissected aortas for 30 min before DHE staining. n = 3. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (C) Representative western blot analysis and quantification of monomer/dimer eNOS ratios in the aortas of WT and FAM3D^−/− mice treated with saline or AngII for 14 days. n = 5. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (D) Representative DAF-FM DA en face staining and quantification of NO in ECs from the thoracic aortas of WT and FAM3D^−/− mice infused with AngII and administered with or without 2,4- DAHP (10 mmol/L) in drinking water for 14 days. n = 5. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (E) SBP was measured by the tail-cuff method in WT and FAM3D^−/− mice infused with AngII and administered with or without DAHP (10 mmol/L) in drinking water for 14 days. n = 4–5. Data are represented as mean ± SEM. Repeated-measures analysis using a mixed-effects model followed by Tukey’s multiple comparisons test, ∗p < 0.05, WT + AngII vs. FAM3D^−/− + AngII; ns, no significance, WT + AngII + DAHP vs. FAM3D^−/− + AngII + DAHP.

Figure 5

FAM3D causes eNOS uncoupling through FPR1/2-mediated oxidative stress (A) Representative western blot and quantification of monomer/dimer eNOS ratios in HUVECs treated with FAM3D (10 nmol/L) for 6 h after preincubation with CsH (1 μmol/L) and/or WRW4 (1 μmol/L) for 2 h. n = 4. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (B) Quantification of intracellular NO by DAF-FM DA staining in human umbilical vein ECs (HUVECs) treated with FAM3D (10 nmol/L) for 6 h after preincubation with CsH (1 μmol/L) and/or WRW4 (1 μmol/L) for 2 h. n = 4. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (C) Quantification of ROS by DCFH-DA staining in HUVECs treated with FAM3D (10 nmol/L) for various times. n = 3. Data are represented as mean ± SEM. One-way ANOVA followed by Dunnett’s multiple comparisons test, ∗p < 0.05. (D) Quantification of ROS by DCFH-DA staining in HUVECs treated with FAM3D (10 nmol/L) for 60 min after preincubation with CsH (1 μmol/L) and/or WRW4 (1 μmol/L) for 2 h. n = 4. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (E) Quantification of O₂⁻ generation by DHE staining in HUVECs treated with FAM3D (10 nmol/L) in the presence or absence of L-NAME (200 μmol/L) for 60 min. n = 3. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (F) Quantification of intracellular NO by DAF-FM DA staining in HUVECs treated with FAM3D (10 nmol/L) for 6 h after preincubation with NAC (5 mmol/L) or apocynin (100 μmol/L) for 4 h. n = 4. Data are represented as mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparisons test, ∗p < 0.05. (G) Representative western blot and quantification of monomer/dimer eNOS radios in HUVECs treated with FAM3D (10 nmol/L) for 6 h after preincubation with NAC (5 mmol/L) or apocynin (100 μmol/L) for 4 h. n = 3. Data are represented as mean ± SEM. Two-way ANOVA followed by Sidak’s multiple comparisons test, ∗p < 0.05.

Figure 6

Endothelial FAM3D knockdown alleviates AngII- and DOCA-salt-induced hypertension in mice (A) Schematic diagram showing the injection of AAV9-Tie2-shRNA (shFAM3D or shControl, 5 × 10¹¹ v.g./mice) via tail veins followed by 14 days of AngII induction in C57BL/6 mice. (B) Representative western blot and quantification of monomer/dimer eNOS ratios and FAM3D in the aortas of C57BL/6 mice infected with AAV9-Tie2-shControl or AAV9-Tie2-shFAM3D followed by AngII infusion for 14 days. n = 5–6. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (C) Representative DAF-FM DA en face staining and quantification of NO in ECs from the thoracic aortas of C57BL/6 mice infected with AAV9-Tie2-shControl or AAV9-Tie2-shFAM3D followed by AngII infusion for 14 days. n = 5. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (D) SBP of C57BL/6 mice infected with AAV9-Tie2-shControl or AAV9-Tie2-shFAM3D followed by AngII infusion for 14 days. n = 5–6. Data are represented as mean ± SEM. Repeated-measures analysis using a mixed-effects model, ∗p < 0.05. (E) Schematic diagram showing the injection of AAV9-Tie2-shRNA (shFAM3D or shControl, 5 × 10¹¹ v.g./mice) via tail veins followed by 21 days of DOCA-salt treatment in C57BL/6 mice. (F) Representative western blot and quantification of monomer/dimer eNOS ratios and FAM3D in the aortas of C57BL/6 mice infected with AAV9-Tie2-shControl or AAV9-Tie2-shFAM3D followed by DOCA-salt treatment for 21 days. n = 5–6. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (G) Representative DAF-FM DA en face staining and quantification of NO in ECs from the thoracic aortas of C57BL/6 mice infected with AAV9-Tie2-shControl or AAV9-Tie2-shFAM3D followed by DOCA-salt treatment for 21 days. n = 4. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (H) SBP of C57BL/6 mice infected with AAV9-Tie2-shControl or AAV9-Tie2-shFAM3D followed by DOCA-salt treatment for 21 days. n = 6. Data are represented as mean ± SEM. Repeated-measures analysis using a mixed-effects model, ∗p < 0.05.

Figure 7

The FAM3D-neutralizing antibodies ameliorate AngII- and DOCA-salt-induced hypertension in mice (A) Schematic diagram showing the administration of the FAM3D-neutralizing antibodies (6D7) or mouse IgG (100 μg/30 g) to C57BL/6 mice during 14 days of AngII infusion. (B) Representative western blot and quantification of monomer/dimer eNOS ratios in the aortas of C57BL/6 mice that were intraperitoneally injected with mouse IgG or 6D7 during AngII infusion for 14 days. n = 5. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (C) Representative DAF-FM DA en face staining and quantification of NO in ECs from the thoracic aortas of C57BL/6 mice that were intraperitoneally injected with mouse IgG or 6D7 during AngII infusion for 14 days. n = 3–4. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test, ∗p < 0.05. (D) SBP of C57BL/6 mice that were intraperitoneally injected with mouse IgG or 6D7 during AngII infusion for 14 days. n = 6–7. Data are represented as mean ± SEM. Repeated-measures analysis using a mixed-effects model, ∗p < 0.05. (E) Schematic diagram showing the administration of the 6D7 or mouse IgG (100 μg/30 g) to C57BL/6 mice during 21 days of DOCA-salt treatment. (F) Representative western blot and quantification of monomer/dimer eNOS ratios in the aortas of C57BL/6 mice that were intraperitoneally injected with mouse IgG or 6D7 during DOCA-salt treatment for 21 days. n = 5. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (G) Representative DAF-FM DA en face staining and quantification of NO in ECs from the thoracic aortas of C57BL/6 mice that were intraperitoneally injected with mouse IgG or 6D7 during DOCA-salt treatment for 21 days. n = 4. Data are represented as mean ± SEM. Unpaired Student’s t test, ∗p < 0.05. (H) SBP of C57BL/6 mice that were intraperitoneally injected with mouse IgG or 6D7 during DOCA-salt treatment for 21 days. n = 6. Data are represented as mean ± SEM. Repeated-measures analysis using a mixed-effects model, ∗p < 0.05.