David D. Kim

Endoplasmic Reticulum Stress Controls M2 Macrophage Differentiation and Foam Cell Formation

Background: The interplay of lipid signaling with macrophage phenotype is critical for vascular disease progression. Results: ER stress links scavenger receptor signaling to macrophage phenotype and foam cell formation through a JNK- and PPARγ-dependent pathway. Conclusion: ER stress is a functional switch controlling macrophage phenotype and cellular cholesterol content. Significance: Suppression of ER stress is a potential therapeutic target to reduce atherosclerosis progression. Macrophages are essential in atherosclerosis progression, but regulation of the M1 versus M2 phenotype and their role in cholesterol deposition are unclear. We demonstrate that endoplasmic reticulum (ER) stress is a key regulator of macrophage differentiation and cholesterol deposition. Macrophages from diabetic patients were classically or alternatively stimulated and then exposed to oxidized LDL. Alternative stimulation into M2 macrophages lead to increased foam cell formation by inducing scavenger receptor CD36 and SR-A1 expression. ER stress induced by alternative stimulation was necessary to generate the M2 phenotype through JNK activation and increased PPARγ expression. The absence of CD36 or SR-A1 signaling independently of modified cholesterol uptake decreased ER stress and prevented the M2 differentiation typically induced by alternative stimulation. Moreover, suppression of ER stress shifted differentiated M2 macrophages toward an M1 phenotype and subsequently suppressed foam cell formation by increasing HDL- and apoA-1-induced cholesterol efflux indicating suppression of macrophage ER stress as a potential therapy for atherosclerosis.

Nitroglycerin-Induced S-nitrosylation and Desensitization of Soluble Guanylyl Cyclase Contribute to Nitrate Tolerance

Nitrates such as nitroglycerin (GTN) and nitric oxide donors such as S-nitrosothiols are clinically vasoactive through stimulation of soluble guanylyl cyclase (sGC), which produces the second messenger cGMP. Development of nitrate tolerance, after exposure to GTN for several hours, is a major drawback to a widely used cardiovascular therapy. We recently showed that exposure to nitric oxide and to S-nitrosothiols causes S-nitrosylation of sGC, which directly desensitizes sGC to stimulation by nitric oxide. We tested the hypothesis that desensitization of sGC by S-nitrosylation is a mechanism of nitrate tolerance. Our results established that vascular tolerance to nitrates can be recapitulated in vivo by S-nitrosylation through exposure to cell membrane-permeable S-nitrosothiols and that sGC is S-nitrosylated and desensitized in the tolerant, treated tissues. We next determined that (1) GTN treatment of primary aortic smooth muscle cells induces S-nitrosylation of sGC and its desensitization as a function of GTN concentration; (2) S-nitrosylation and desensitization are prevented by treatment with N-acetyl-cysteine, a precursor of glutathione, used clinically to prevent development of nitrate tolerance; and (3) S-nitrosylation and desensitization are reversed by cessation of GTN treatment. Finally, we demonstrated that in vivo development of nitrate tolerance and crosstolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues. These results suggest that in vivo nitrate tolerance is mediated, in part, by desensitization of sGC through GTN-dependent S-nitrosylation.

Acupuncture Reduces Experimental Renovascular Hypertension Through Mechanisms Involving Nitric Oxide Synthases

Objective: To test the hypothesis that acupuncture on stomach 36 point (ST‐36) reduces hypertension by activating nitric oxide synthase signaling mechanisms.

Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability

The molecular mechanisms of endothelial nitric oxide synthase (eNOS) regulation of microvascular permeability remain unresolved. Agonist-induced internalization may have a role in this process. We demonstrate here that internalization of eNOS is required to deliver NO to subcellular locations to increase endothelial monolayer permeability to macromolecules. Using dominant-negative mutants of dynamin-2 (dyn2K44A) and caveolin-1 (cav1Y14F), we show that anchoring eNOS-containing caveolae to plasma membrane inhibits hyperpermeability induced by platelet-activating factor (PAF), VEGF in ECV-CD8eNOSGFP (ECV-304 transfected cells) and postcapillary venular endothelial cells (CVEC). We also observed that anchoring caveolar eNOS to the plasma membrane uncouples eNOS phosphorylation at Ser-1177 from NO production. This dissociation occurred in a mutant- and cell-dependent way. PAF induced Ser-1177-eNOS phosphorylation in ECV-CD8eNOSGFP and CVEC transfected with dyn2K44A, but it dephosphorylated eNOS at Ser-1177 in CVEC transfected with cav1Y14F. Interestingly, dyn2K44A eliminated NO production, whereas cav1Y14F caused reduction in NO production in CVEC. NO production by cav1Y14F-transfected CVEC occurred in caveolae bound to the plasma membrane, and was ineffective in causing an increase in permeability. Our study demonstrates that eNOS internalization is required for agonist-induced hyperpermeability, and suggests that a mechanism by which eNOS is activated by phosphorylation at the plasma membrane and its endocytosis is required to deliver NO to subcellular targets to cause hyperpermeability.

Internalization of eNOS via caveolae regulates PAF-induced inflammatory hyperpermeability to macromolecules

Endothelial nitric oxide (NO) synthase (eNOS) is thought to regulate microvascular permeability via NO production. We tested the hypotheses that the expression of eNOS and eNOS endocytosis by caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial permeability to macromolecules. We used bovine coronary postcapillary venular endothelial cells (CVECs) because these cells are derived from the microvascular segment responsible for the transport of macromolecules in inflammation. We stimulated CVECs with platelet-activating factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production, and CVEC monolayer permeability to FITC-dextran 70 KDa (Dx-70). PAF translocated eNOS from plasma membrane to cytosol, induced changes in the phosphorylation state of the enzyme, and increased NO production from 4.3+/-3.8 to 467+/-22.6 nM. PAF elevated CVEC monolayer permeability to FITC-Dx-70 from 3.4+/-0.3 x 10(-6) to 8.5+/-0.4 x 10(-6) cm/s. The depletion of endogenous eNOS with small interfering RNA abolished PAF-induced hyperpermeability, demonstrating that the expression of eNOS is required for inflammatory hyperpermeability responses. The inhibition of the caveolar internalization by blocking caveolar scission using transfection of dynamin dominant-negative mutant, dyn2K44A, inhibited PAF-induced hyperpermeability to FITC-Dx-70. We interpret these data as evidence that 1) eNOS is required for hyperpermeability to macromolecules and 2) the internalization of eNOS via caveolae is an important mechanism in the regulation of endothelial permeability. We advance the novel concept that eNOS internalization to cytosol is a signaling mechanism for the onset of microvascular hyperpermeability in inflammation.

Soluble guanylyl cyclase is a target of angiotensin II-induced nitrosative stress in a hypertensive rat model

Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.

Functional Significance of Cytosolic Endothelial Nitric-oxide Synthase (eNOS) REGULATION OF HYPERPERMEABILITY

Endothelial NOS (eNOS)-derived NO is a key factor in regulating microvascular permeability. We demonstrated previously that eNOS translocation from the plasma membrane to the cytosol is required for hyperpermeability. Herein, we tested the hypothesis that eNOS activation in the cytosol is necessary for agonist-induced hyperpermeability. To study the fundamental properties of endothelial cell monolayer permeability, we generated ECV-304 cells that stably express cDNA constructs targeting eNOS to the cytosol or plasma membrane. eNOS-transfected ECV-304 cells recapitulate the eNOS translocation and permeability properties of postcapillary venular endothelial cells (Sánchez, F. A., Rana, R., Kim, D. D., Iwahashi, T., Zheng, R., Lal, B. K., Gordon, D. M., Meininger, C. J., and Durán, W. N. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 6849–6853). We used platelet-activating factor (PAF) as a proinflammatory agonist. PAF activated eNOS by increasing phosphorylation of Ser-1177 and inducing dephosphorylation of Thr-495, increasing NO production, and elevating permeability to FITC-dextran 70 in monolayers of cells expressing wild-type and cytosolic eNOS. PAF failed to increase permeability to FITC-dextran 70 in monolayers of cells transfected with eNOS targeted to the plasma membrane. Interestingly, this occurred despite eNOS Ser-1177 phosphorylation and production of comparable amounts of NO. Our results demonstrate that the presence of eNOS in the cytosol is necessary for PAF-induced hyperpermeability. Our data provide new insights into the dynamics of eNOS and eNOS-derived NO in the process of inflammation.

RAPAMYCIN INHIBITS VEGF-INDUCED MICROVASCULAR HYPERPERMEABILITY IN VIVO

Microcirculation (2010) 17, 1–9. doi: 10.1111/j.1549‐8719.2009.00012.x

Microvascular transport is associated with TNF plasma levels and protein synthesis in postischemic muscle

To better understand the mechanisms of ischemia-reperfusion (I/R) injury, we tested the hypothesis that protein synthesis is involved in the production of tumor necrosis factor (TNF) and in the microvascular transport changes in I/R. To evaluate the hypothesis, we inhibited protein synthesis with topically applied actinomycin D (AMD), measured I/R-induced changes in microvascular transport, and bioassayed the venous plasma levels of TNF. The rat cremaster muscle I/R model consisted of 4 h of ischemia followed by 2 h of reperfusion. Changes in transport were determined by integrated optical intensity (IOI) using FITC-Dextran 150 as tracer. Animals were separated into four groups: 1) control (C), 2) control treated with AMD (C + AMD), 3) I/R, and 4) I/R treated with AMD (I/R + AMD). The mean (+/-SE) maximal IOI in C and C + AMD were 3.0 +/- 1.0 and 3. 7 +/- 0.7 units, respectively. I/R elevated mean maximal IOI to 21.8 +/- 1.9 units (P < 0.05 vs. C, C + AMD, I/R + AMD). Treatment with AMD reduced the I/R-induced mean maximal IOI to 9.7 +/- 2.0 units (P < 0.05 vs. I/R). In I/R group, plasma TNF levels increased (relative to preischemia baseline) immediately after the release of the vascular occlusion to 250 pg/ml and reached a peak value of 342 pg/ml at 60 min of reperfusion. In the I/R + AMD group, AMD reduced TNF increase to 44 pg/ml. The C and C + AMD groups showed no differences in TNF values during the 6 h of observation. We conclude that protein synthesis and TNF generation are at least partially involved in I/R-induced changes in microvascular transport.To better understand the mechanisms of ischemia-reperfusion (I/R) injury, we tested the hypothesis that protein synthesis is involved in the production of tumor necrosis factor (TNF) and in the microvascular transport changes in I/R. To evaluate the hypothesis, we inhibited protein synthesis with topically applied actinomycin D (AMD), measured I/R-induced changes in microvascular transport, and bioassayed the venous plasma levels of TNF. The rat cremaster muscle I/R model consisted of 4 h of ischemia followed by 2 h of reperfusion. Changes in transport were determined by integrated optical intensity (IOI) using FITC-Dextran 150 as tracer. Animals were separated into four groups: 1) control (C), 2) control treated with AMD (C + AMD), 3) I/R, and 4) I/R treated with AMD (I/R + AMD). The mean (±SE) maximal IOI in C and C + AMD were 3.0 ± 1.0 and 3.7 ± 0.7 units, respectively. I/R elevated mean maximal IOI to 21.8 ± 1.9 units ( P < 0.05 vs. C, C + AMD, I/R + AMD). Treatment with AMD reduced the I/R-induced mean maximal IOI to 9.7 ± 2.0 units ( P< 0.05 vs. I/R). In I/R group, plasma TNF levels increased (relative to preischemia baseline) immediately after the release of the vascular occlusion to 250 pg/ml and reached a peak value of 342 pg/ml at 60 min of reperfusion. In the I/R + AMD group, AMD reduced TNF increase to 44 pg/ml. The C and C + AMD groups showed no differences in TNF values during the 6 h of observation. We conclude that protein synthesis and TNF generation are at least partially involved in I/R-induced changes in microvascular transport.

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.