A, Representative microscopy images of the migration of monocytes from hypertensive patients (upper panels) compared with normotensive control subjects (lower panels) in the absence (left panels) or in the presence (right panels) of 2-APB (100 µmol/L). Expreriments were performed in triplicate. Migration was induced by chemoattractant fMLP (100 nm/L). Magnification 4×, bar denotes 100 µm. B, C, Summary data showing fMLP-induced or TNF-α-induced migration of monocytes from normotensive control subjects (NT; open bars) and hypertensive patients (HT, filled bars) in the absence or presence of 2-APB (100 µmol/L). After the incubation time the polycarbonate filter membranes were dehydrated, stained using fura2-AM and fluorescence was detected at 510 nm emission with 360 nm excitation. Migration rates were normalized to the mean migration rate of monocytes maintained in the medium condition (control). We observed an increased fMLP-induced or TNF-α-induced migration of monocytes from patients with essential hypertension. *p<0.05, **p<0.01 compared to chemoattractant alone in normotensive control subjects, +p>0.05 for the comparison in the presence of 2-APB NT vs. HT. Each n = 10 or 11. D; Spontaneous migrations of monocytes from normotensive control subjects (NT; open bars) and hypertensive patients (HT, filled bars) were tested using medium or in the presence of 2-APB (100 µmol/L). The data was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. P>0.05 compared to normotensive control subjects. Data are percent of medium as mean ± SEM of three independent experiments.

A, B; Representative fluorescence tracings in monocytes from normotensive control subjects (A) and hypertensive patients (B) after administration of fMLP (100 nm/L) in the absence and presence of TRP channel-inhibitor 2-APB. C; Summary data of changes of intracellular calcium concentration in monocytes from normotensive control subjects (NT; open bars) and patients with essential hypertension (HT; filled bars) by several doses of fMLP (10 nmol/L, 50 nmol/L, and 100 nm/L). Each n = 10; *p<0.05 for the comparison HT vs. NT. D, E; fMLP-induced cation influx into monocytes indicated by quenching of fura-2 by manganese (Mn²⁺, 1 µmol/L). Representative raw data, showing mamganese quenching with a stimulus (fMLP, 100 nmol/L) or without stimulus (control, w/o fMLP) in monocytes from normotensive control subjects (D; open black or grey circle; NT) and patients with essential hypertension (E; filled black or grey circle; HT). F; Bar graph shows the summary data of manganese quenching rate obtained from normotensive control subjects (NT, n = 6) and patients with essential hypertension (HT, n = 8) under the control conditions or after administration of fMLP (100 nmol/L). *p<0.05 or **p<0.01 for the comparison with their controls; and #p<0.05 or ## p<0.01 for the comparison HT (filled bars) vs. NT (open bars).

A; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT) and patients with essential hypertension (HT) in the absence or presence of TRPC3 antigens (TRPC3+Ag). The predicted molecular weight of TRPC3 is 97 kDa. B; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT, n = 8), patients with type 2 diabetes mellitus (DM, n = 9), patients with essential hypertension (HT, n = 8) or hypertensive patients with type 2 diabetes mellitus (HT+DM, n = 10). Summary data of the TRPC3 expression (normalized to GAPDH). *p<0.05, compared to NT. Data are mean ± SEM. C; Representative in-cell western assay and summary data of the TRPC3 expression (normalized to CD14 expression used as an internal reference) in monocytes from normotensive control subjects (Normotensive, and opened bars, n = 3) and patients with essential hypertension (Hypertensive, filled bars, n = 3) under control conditions and after transfection with scrambled siRNA or specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 (visible in green) normalized to CD14 (used as an internal reference). Measurements were performed in duplicate for each sample. *p<0.05 or **p<0.01 for the comparison with their controls; and ## p<0.01 for the comparison Hypertensive (filled bars) vs. Normotensive (open bars). D; Representative in-cell western assay and summary data of the TRPC3 and TRPC6 expression in monocytes from normotensive control subjects under control conditions and after transfection with specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 and TRPC6 expression (visible in green) normalized to CD14 (visible in red used as an internal reference). Measurements were performed in duplicate for each sample. **p<0.01 compared to control conditions. Data are mean ± SEM of three independent experiments. E; Summary data of the fMLP-induced monocyte migration from hypertensive patients (HT, filled bars) and normotensive control subjects (NT, opened bars) quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. Monocytes chemotaxis was expressed as the mean number of migrated cells per high-power fields from duplicate wells. Experiments were performed under control conditions, after transfection with scrambled siRNA or specific siRNA against TRPC3. *p<0.05; **p<0.01 compared to normotensive control subjects under control conditions. Data are mean ± SEM of eight independent experiments. F; Spontaneous migrations of monocytes from normotensive control subjects (NT; open bars) and hypertensive patients (HT, filled bars) were tested using medium or after transfection with scrambled siRNA or specific siRNA against TRPC3. The data was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. P>0.05 compared to NT. Data are percent of medium as mean ± SEM of three independent experiments.

A, Peripheral blood monocytes subpopulations were analyzed by flow-cytometry. After labeling with anti-CD14 phycoerythrin (PE) conjugated and anti-CD16 FITC conjugated, monocytes from patients with essential hypertension (HT, filled bars) and normotensive control subjects (NT; open bars) were readily separated into three distinct subsets according to CD14 and CD16 positivity. Data are mean ± SEM, each n = 11, P>0.05 NT vs. HT. B, Expression of fMLP receptors using immunoblotting with specific antibodies. The data showed that fMLP receptors were not significantly different between in monocytes from patients with essential hypertension (HT, filled bars) and normotensive control subjects (NT; open bars). Data are mean ± SEM, n = 6, P>0.05 NT vs. HT. C, D, Representative micrographs of fMLP-induced polarization response of monocytes (C). Summary data of fMLP induced polarization response of monocytes from healthy control subjects (NT; open bars) and from patients with hypertension (HT, filled bars). Data are mean ± SEM, **p<0.01 compared to NT fMLP-stimulation (0 min); ##p<0.01 compared to HT fMLP-stimulation (0 min); P>0.05 NT vs. HT; each n = 12 (D).

A; Summary data of fMLP-induced monocytes migration was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. Monocytes chemotaxis was expressed as the mean number of migrated cells per 40× fields from duplicate wells. Experiments were performed under control conditions (fMLP, n = 6), in the presence of genistein (Geni, n = 6) or wortmannin (Wort, n = 6) and PD98059 (PD, n = 3). Data are mean ± SEM of three to six independent experiments. **p<0.01 compared to their chemoattractant (fMLP) alone; # p<0.05 for comparison HT (filled bars) vs. NT (open bars).

A, B; fMLP activates ERK or phosphorylation of ERK (A) and Akt or phosphorylation of Akt (B) in a dose- and time-dependent manner in monocytes from normotensive control subjects. 10 nmol/L open bars, 100 nmol/L filled bars. Data are mean ± SEM, n = 3. *p<0.05 compared to lower concentration conditions. C, D; Increased fMLP-induced phosphorylation of ERK (C) and Akt (D) in monocytes from patients with essential hypertension. The proteins were measured using immunoblotting with specific antibodies. Data are mean ± SEM from three independent experiments. *p<0.05 compared to normotensive control subjects. E; fMLP activates monocytes by an ERK-dependent and Akt-dependent pathway. Akt, ERK, or pERK and pAkt were measured using immunoblotting with specific antibodies. In the presence of 2-APB or after administration of specific siRNA against TRPC3, the fMLP-induced ERK, pERK; Akt and pAkt were significantly reduced when compared with control conditions. Data are mean ± SEM from six independent experiments. *p<0.05; **p<0.01 compared to control.