RESUMEN
Matrix metalloproteinases (MMPs) are endopeptidases, and their activity depends on calcium and zinc metal ions. These enzymes are expressed originally in zymogenic form, where the active site of proteins is closed by a prodomain which is removed during activation. A homeostatic balance of their activity is primarily regulated by a 'cysteine switch' located on a consensus sequence of the prodomain and natural endogenous inhibitors, called tissue inhibitors of metalloproteinases (TIMPs). Breakage of this homeostasis may lead to various pathological conditions, which may require further activation and/or inhibition of these enzymes to regenerate that balance. Here, we report four modulators, more specifically, three inhibitors (I1, I2 and I3), and one exogenous activator (L) of the active form of human collagenase MMP-1 (without prodomain). The results were confirmed by binding studies using fluorescence-based enzyme assays.
RESUMEN
Angiotensin II (Ang II) modulates blood pressure and atherosclerosis development through its vascular type-1 (AT1R) and type-2 (AT2R) receptors, which have opposing effects. AT2R activation produces hypotension, and is anti-atherogenic. Targeted overexpression of AT2Rs in vascular smooth muscle cells (VSMCs) indicates that these effects are due to increased nitric oxide (NO) generation. However, the role of endogenous VSMC AT2Rs in these events is unknown. Effect of 7-day low-dose Ang II-infusion (12 µg/kg/hr) on blood pressure was tested in 9-week-old apoE((-/-)) mice fed a low or high cholesterol diet (LCD or HCD, respectively). Cardiac output was measured by echocardiography. Immunohistochemistry was performed to localize and quantify AT2Rs and p-Ser(1177)-endothelial nitric oxide synthase (eNOS) levels in the aortic arch. PD123319 and GW-9662 were used to selectively block the AT2R and peroxisome proliferator-activated receptor-γ (PPAR-γ), respectively. Ang II infusion decreased blood pressure by 12 mmHg (P < 0.001) in LCD/apoE((-/-)) mice without altering cardiac output; a response blocked by PD123319. Although, AT2R stimulation neither activated eNOS (p-Ser(1177)-eNOS) nor changed plasma NO metabolites, it caused an ~6-fold increase in VSMC PPAR-γ levels (P < 0.001) and the AT2R-mediated hypotension was abolished by GW-9662. AT2R-mediated hypotension was also inhibited by HCD, which selectively decreased VSMC AT2R expression by ~6-fold (P < 0.01). These findings suggest a novel pathway for the Ang II/AT2R-mediated hypotensive response that involves PPAR-γ, and is down regulated by a HCD.