DAPK1 acts as a positive regulator of hypertension via induction of vasoconstriction

Figure 1:. Death-associated protein kinase 1 (DAPK1) suppression lowers blood pressure and reduces vascular and organ damage in spontaneously hypertensive rats (SHRs). (A–C) Tail-cuff plethysmography: systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP). (D,E) Ultrasound: pulse wave velocity (PWV) and aortic wall thickness. (F) Hematoxylin-eosin (H&E)-stained abdominal aorta sections (400× magnification). (G–J) immunohistochemical (IHC) analysis: proliferating cell nuclear antigen (PCNA) (G,H) and DAPK1 (I,J) expression in aortic tissues (scale bar = 50 µm). (K,L) Echocardiography: left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF). (M,N) H&E-stained cardiac and renal tissues (scale bar = 50 µm). n=7, *,#p<0.05 vs. sh-Control and sh-DAPK1, respectively.

Figure 2:. DAPK1 deficiency alleviates angiotensin II (Ang II)-induced hypertension and vascular pathology. (A–C) SBP, DBP, and MAP in each group. (D,E) Ultrasonography: PWV and aortic wall thickness. (F) H&E-stained cross-sections of abdominal aorta tissues (scale bar = 50 µm). (G,H) IHC analysis: PCNA expression in aortic tissues (scale bar = 50 µm). (I,J) LVFS and LVEF. (K,L) H&E-stained cardiac and renal tissues (scale bar = 50 µm). n=5, *,#p<0.05 vs. DAPK1+/+ + Ang II and DAPK1−/− +Ang II, respectiely.

Figure 3:. DAPK1 deficiency mitigates Ang II-provoked aortic constriction in hypertensive mice. (A) Volcano plots: differentially expressed transcripts (DETs) in abdominal aorta tissues (p<0.05, |fold change| ≥ 2). (B,C) Comparative transcript analysis: increased and decreased DETs in DAPK1+/++ Ang II vs. DAPK1+/+ + saline groups and DAPK1−/− + Ang II vs. DAPK1+/+ + Ang II. (D–F) KEGG analysis: Top 30 enriched pathways in DAPK1+/+ + Ang II vs. DAPK1+/+ + saline (D) and DAPK1−/− + Ang II vs. DAPK1+/+ + Ang II (E), with integrated pathway overlaps (F). (G) Immunofluorescence staining: F-actin (red) with DAPI-labeled nuclei (blue); scale bar = 20 µm. (H) Effect of DAPK1 knockdown on tension in Ang II pre-constricted abdominal aorta rings.

Figure 4:. DAPK1 inhibition reverses Ang II-induced hypertension and vascular damage. (A–C) TC-DAPK6 reduces SBP, DBP, and MAP in Ang II-triggered hypertensive mice. (D,E) Ultrasonography: PWV and aortic wall thickening. (F) H&E-stained cross-sections of the abdominal aorta. (G,H) IHC analysis: PCNA expression in aortic tissues (scale bar = 50 µm). (I,J) LVFS and LVEF measurements. (K,L) H&E-stained cardiac and renal tissues. (M–P) IHC analysis: DAPK1 (M) and p-DAPK1(S308) (O) expression in aortic tissues, with statistical representation. n=6,*,#p<0.05 vs. Control and Ang II + ddH₂O, respectively.

Figure 5:. Ang II increases intracellular calcium and activates DAPK1 in vascular smooth muscle cells (VSMCs). (A,B) Real-time confocal microscopy: VSMCs loaded with Fluo-4 showing intracellular Ca²+ increase after Ang II (1 μM) stimulation (200× magnification). Ca²+ levels were normalized to baseline. (C,D) Western blotting: p-DAPK1(S308)/DAPK1 ratio over time (*P<0.05 vs. 0 min). (E,F) Western blot analysis was performed to determine the ratio protein expression of p-DAPK1(S308)/DAPK1 protein expression, *p<0.05 vs. the Control group, #p<0.05 vs. Ang II + group.

Figure 6:. DAPK1 lowers blood pressure by inhibiting myosin light chain (MLC)-mediated vasoconstriction. IHC analysis: Phosphorylated MLC at Ser19 (p-MLC(S19)) and total MLC in abdominal aortic tissues. (A–D) p-MLC(S19) and MLC levels in SHR (n=7, *,#p<0.05 vs. sh-Control and sh-DAPK1, respectively). (E–H) DAPK1+/+ and DAPK1−/− mice treated with Ang II (n=5, *,#p<0.05 vs. DAPK1+/+ + Ang II and DAPK1−/− + Ang II, respectively). (I–L) Control vs. Ang II + ddH₂O (n=6, *,#p<0.05 vs. Control and Ang II, respectively); 400× magnification, scale bar = 50 μm.