Homocysteine impairs endothelial wound healing by activating metabotropic glutamate receptor 5

Abstract

Objective: Hcy is an independent risk factor for cerebrovascular disease and cognitive impairment. The purpose of this study was to elucidate the role of mGluR5 in Hcy-mediated impairment of cerebral endothelial wound repair.

Methods: Mouse CMVECs (bEnd.3) were used in conjunction with directed pharmacology and shRNA. AutoDock was used to simulate the docking of ligand-receptor interactions.

Results: Hcy (20 μM) significantly increased Cx43-pS368 by mGluR5- and PKC-dependent mechanisms. Hcy attenuated wound repair by an mGluR5-dependent mechanism over the six-day study period but did not alter cell proliferation in a proliferation assay, suggesting that the attenuation of wound repair may be due to dysfunctional migration in HHcy. Hcy increased the expression of Cx43 and Cx43-pS368 at the wound edge by activating mGluR5. Direct activation of mGluR5, using the specific agonist CHPG, was sufficient to reproduce the results whereas KO of mGluR5 with shRNA, or inhibition with MPEP, blocked the response to Hcy.

Conclusions: Inhibition of mGluR5 activation could be a novel strategy for promoting endothelial wound repair in patients with HHcy. Activation of mGluR5 may be a viable strategy for disrupting angiogenesis.

Figure 1.

Hcy increases Cx43 phosphorylation on S368 (Cx43-pS368). (A) Cx43-pS368 (green) and total Cx43 (red) expression after treating with Hcy for the indicated times; control is secondary antibody only (omission of the primary antibody). Scale bar is 80 μm and applies to all images. (B-C) ELISA for the expression of Cx43 (B) and Cx43-pS368 (C) (n= 10). (D) Two hour treatments with Hcy and PMA, an activator of PKC, induce significantly greater expression of Cx43-pS368 (n = 10).

Figure 2.

Hcy elevates Cx43-pS368 via PKC and metabotropic glutamate receptor 5 (mGluR5). (A-B) Two-hour treatments with Hcy alone or in combinations with PKC or mGluR5 inhibition do not alter Cx43 expression (n = 10). (C) Two-hour treatments with 20 μM Hcy and 250 nM PMA induce Cx43-pS368, which is blocked by PKC inhibition (1 μM BIM) (n = 8–14). BIM alone does not affect Cx43-pS368. (D) Twenty-micromolar Hcy treatment significantly elevates Cx43-pS368, which is rescued by treating with 25 or 100 μM MPEP, an antagonist of mGluR5 (n = 5–10). MPEP alone does not alter the Cx43-pS368 response (data not shown).

Figure 3.

mGluR5 activation induces Cx43-pS368 via PKC. (A-B) Two-hour treatment with CHPG, mGluR5 agonist, and BIM, PKC inhibitor, do not alter Cx43 expression (n = 10). (C) Two-hour treatments with CHPG dose-dependently raises Cx43-pS368 expression (n = 9–12). (D) About 50 and 100 μM CHPG increases Cx43-pS368 expression, which is blocked by PKC inhibition (1 μM BIM) (n = 8–10), suggesting that mGluR5 activation raises Cx43-pS368 via PKC

Figure 4.

AutoDock predicts docked energies and ligand binding sites on mGluR5. AutoDock, a computer-based modeling software program, was used to predict mGluR5-binding sites and calculate docked energies for (A) glutamate, (B) CHPG (a selective agonist), and (C) Hcy. Glutamate and CHPG are known to bind mGluR5, demonstrating the accuracy of the modeling program in our hands. The binding sites of mGluR5 for Hcy are in the same orthosteric pocket as the two known agonists, glutamate and CHPG. The docked energy for Hcy is intermediate between mGluR5 and CHPG, which suggests that Hcy is likely to bind mGluR5.

Figure 5.

Hcy impairs EC wound repair but not proliferation. (A) About 20 and 200 μM Hcy significantly attenuates daily wound repair distance in in vitro scrape model (n = 6). (B) Hcy dose-dependently impairs the wound repair rate for the duration of one to six days (n = 6). (C-D) Single-cell suspension was plated at 20–30% confluence. The following morning, cells were treated with Hcy, 18βGA, or vehicle control for three days (n = 10). Data are the increase from values determined in parallel cultures harvested at the time of treatment (morning after plating). Cell number (C) and total protein content (D) were not affected by Hcy but were significantly decreased by 18βGA treatment. (E) To better understand the effect of 18βGA, proliferation experiments were repeated with daily measurements in the context of continuous treatment (first six columns) and with treatment for 24 hours only beginning at each of the three days of study (last three columns). Bars with different letters (a-d) are significantly different from one another (p <0.05). Although 18βGA reduced proliferation, the effect appears to be blocking of one day’s worth of proliferation regardless of the timing or duration of treatment (n = 10).

Figure 6.

Hcy impairs EC wound repair via mGluR5. Hcy (20 μM) significantly reduces EC (A) daily repair distance and (B) repair rate for one to six days, which are rescued by treatment with the mGluR5-selective antagonist MPEP (25 μM) (n = 3). CHPG, a mGluR5-specific agonist, dose-dependently impairs (C) EC repair distance within each day (n = 3) and (D) EC repair rate for one- to six- day observations (n = 3).

Figure 7.

Stable KO of mGluR5 using shRNA rescues the wound repair inhibiting effects of Hcy. Chronic treatment with Hcy (20 μM) impaired the daily wound repair distance (A) and six-day repair rate (B) in cells with an NS control shRNA, which was rescued by shRNA KO of mGluR5 to levels observed in both cell types without Hcy treatment (n = 3). A subset of NS cells (“switch”) was treated with Hcy for five days followed by change to normal medium (no Hcy) for the final day, resulting in a rescued rate (slope of line) to levels observed in the other cell lines. (C-D) Treatment with 18βGA reduced the wound repair distance at each day (C) and the overall six-day repair rate (D) but these effects were entirely due to the nearly complete arrest of repair during the first 24 hours following wounding/treatment because the slope of the daily repair was identical beyond the first day (n = 6).

Figure 8.

Hcy increases the expression of Cx43 and its phosphorylation at S368 expression via mGluR5 during wound repair. (A) ELISA results demonstrate that Hcy (20 μM) significantly increases Cx43 expression, which is blocked by mGluR5 KO with shRNA but not NS control shRNA (n = 7; *p < 0.05 vs. KO) at 24 and 72 hours after wounding. (B) ELISA results further demonstrate that Hcy significantly induces the phosphorylation of Cx43 at S368, which is blocked by mGluR5 KO with shRNA but not NS control shRNA (n = 7; *p < 0.05 vs. KO) at 24 and 72 hours after wounding.

Figure 9.

Hcy induces increased expression of Cx43 (red) and Cx43-pS368 (green; yellow is overlap of red and green) along the edge of wounded ECs at 24 and 72 hours after scrape wounding. Wound edges are toward the center line of the paired images and are indicated by dashed lines (i.e., to the right of control images and to the left of Hcy images). Scale bar is 100 μm and applies to all images.