Rapamycin inhibits VEGF-induced microvascular hyperpermeability in vivo.

OBJECTIVE: To test the hypothesis that rapamycin inhibits induced microvascular hyperpermeability directly in vivo. METHODS: Male golden Syrian hamsters (80-120 g) were treated with either rapamycin (at 0.1, 0.5, 2, and 10 mg/kg i.p.) or vehicle at 24 hours and at 1 hour prior to preparation of the cheek pouch. Caveolin-1 scaffolding (1 mg/kg; positive inhibitory control) was injected i.p. 24 hours prior to the experiment. 10(-8) M vascular endothelial growth factor (VEGF) or 10(-7) M platelet-activating factor (PAF) were topically applied to the cheek pouch. Microvascular permeability and arteriolar diameter were assessed using integrated optical intensity (IOI) and vascular wall imaging, respectively. RESULTS: Rapamycin at 0.1 and 0.5 mg/kg significantly reduced VEGF-stimulated mean IOI from 63.0 +/- 4.2 to 9.7 +/- 5.0 (85% reduction, P < 0.001) and 3.6 +/- 2.7 (95% reduction, P < 0.001), respectively. Rapamycin at 2 mg/kg also lowered VEGF-stimulated hyperpermeability (40% reduction, P < 0.05). However, 10 mg/kg rapamycin increased VEGF-induced microvascular hyperpermeability. Rapamycin at 0.5 mg/kg attenuated VEGF-induced vasodilation and PAF-induced hyperpermeability, but did not inhibit PAF-induced vasoconstriction. CONCLUSIONS: At therapeutically relevant concentrations, rapamycin inhibits VEGF- and PAF-induced microvascular permeability. This inhibition is (i) a direct effect on the endothelial barrier, and (ii) independent of arteriolar vasodilation. Rapamycin at 10 mg/kg stimulates effectors that increase microvascular permeability.

Independent regulation of periarteriolar and perivenular nitric oxide mechanisms in the in vivo hamster cheek pouch microvasculature.

OBJECTIVE: We tested the hypothesis that differential stimulation of nitric oxide (NO) production can be induced in pre- and postcapillary segments of the microcirculation in the hamster cheek pouch. MATERIALS AND METHODS: We applied acetylcholine (ACh) or platelet-activating factor (PAF) topically and measured perivascular NO concentration ([NO]) with NO-sensitive microelectrodes in arterioles and venules of the hamster cheek pouch. We also measured NO in cultured coronary endothelial cells (CVEC) after ACh or PAF. RESULTS: ACh increased periarteriolar [NO] significantly in a dose-dependent manner. ACh at 1 microM increased [NO] from 438.1+/-43.4 nM at baseline to 647.9+/-66.3 nM, while 10 microM of ACh increased [NO] from baseline to 1,035.0+/-59.2 nM (P<0.05). Neither 1 nor 10 microM of ACh changed perivenular [NO] in the hamster cheek pouch. PAF, at 100 nM, increased perivenular [NO] from 326.6+/-50.8 to 622.8+/-41.5 nM. Importantly, 100 nM of PAF did not increase periarteriolar [NO]. PAF increased [NO] from 3.6+/-2.1 to 455.5+/-19.9 in CVEC, while ACh had no effect. CONCLUSIONS: We conclude that NO production can be stimulated in a differential manner in pre- and postcapillary segments in the hamster cheek pouch. ACh selectively stimulates the production of NO only in arterioles, while PAF stimulates the production of NO only in venules.

Endothelial nitric oxide synthase is a molecular vascular target for the Chinese herb Danshen in hypertension.

Danshen, a Chinese herb, reduces hypertension in Oriental medicine. We hypothesized that Danshen acts partially through endothelial nitric oxide synthase (eNOS) signaling mechanisms. We tested the hypothesis using tanshinone II(A), an active ingredient of Danshen, and the two-kidney, one-clip renovascular hypertension model in hamsters. Oral tanshinone (50 microg/100 g body wt) reduced mean arterial pressure (MAP) from 161.2 +/- 6.9 to 130.0 +/- 7.8 mmHg (mean +/- SE; P < 0.05) in hypertensive hamsters. MAP in sham-operated hamsters was 114.3 +/- 9.2 mmHg. Topical tanshinone at 1 microg/ml and 5 microg/ml increased normalized arteriolar diameter from 1.00 to 1.25 +/- 0.08 and 1.57 +/- 0.11, respectively, and increased periarteriolar nitric oxide concentration from 87.1 +/- 11.3 to 146.9 +/- 23.1 nM (P < 0.05) at 5 microg/ml in hamster cheek pouch. N(G)-monomethyl-L-arginine inhibited tanshinone-induced vasodilation. Hypertension reduced eNOS protein relative to sham-operated control. Tanshinone prevented the hypertension-induced reduction of eNOS and increased eNOS expression to levels higher than sham-operated control in hamster cheek pouch. Topical tanshinone increased normalized arteriolar diameter from 1.0 to 1.47 +/- 0.08 in the cremaster muscle of control mice and to 1.12 +/- 0.13 in cremasters of eNOS knockout mice. In ECV-304 cells transfected with eNOS-green fluorescent protein, tanshinone increased eNOS protein expression 1.35 +/- 0.05- and 1.85 +/- 0.07-fold above control after 5-min and 1-h application, respectively. Tanshinone also increased eNOS phosphorylation 1.19 +/- 0.07- and 1.72 +/- 0.20-fold relative to control after 5-min and 1-h application. Our data provide a basis to understand the action of a Chinese herb used in alternative medicine. We conclude that eNOS stimulation is one mechanism by which tanshinone induces vasodilation and reduces blood pressure.