Louis Joseph Ignarro

Significance: Our studies demonstrate that the regulation of endothelial cellular senescence emerges as an additional mechanism underlying the antiatherogenic properties of liver X receptor (LXR) agents. Furthermore, our data suggest that combination therapy with LXR agents and metformin may provide a clinically useful therapeutic strategy to alleviate an LXR activation-mediated adverse effect on liver triglyceride metabolism. The beneficial effects of LXR activation appear to be reduced reactive oxygen species levels and increased endothelial NO synthase activity, both of which will lead to increased NO actions. Among the elderly population, atherosclerosis is a growing problem, leading to an increased risk of mortality by cardiovascular events. Adoption of combination therapy with LXR agents and widely prescribed metformin may be of clinical significance.

Tumor growth requires neoangiogenesis. VEGF is the most potent proangiogenic factor. Dysregulation of hypoxia-inducible factor (HIF) or cytokine stimuli such as those involving the chemokine receptor 4/stromal-derived cell factor 1 (CXCR4/SDF-1) axis are the major cause of ectopic overexpression of VEGF in tumors. Although the CXCR4/SDF-1 pathway is well characterized, the transcription factors executing the effector function of this signaling are poorly understood. The multifunctional Yin Yang 1 (YY1) protein is highly expressed in different types of cancers and may regulate some cancer-related genes. The network involving CXCR4/YY1 and neoangiogenesis could play a major role in cancer progression. In this study we have shown that YY1 forms an active complex with HIF-1α at VEGF gene promoters and increases VEGF transcription and expression observed by RT-PCR, ELISA, and Western blot using two different antibodies against VEGFB. Long-term treatment with T22 peptide (a CXCR4/SDF-1 inhibitor) and YY1 silencing can reduce in vivo systemic neoangiogenesis ( P < 0.01 and P < 0.05 vs. control, respectively) during metastasis. Moreover, using an in vitro angiogenesis assay, we observed that YY1 silencing led to a 60% reduction in branches ( P < 0.01) and tube length ( P < 0.02) and a 75% reduction in tube area ( P < 0.001) compared with control cells. A similar reduction was observed using T22 peptide. We demonstrated that T22 peptide determines YY1 cytoplasmic accumulation by reducing its phosphorylation via down-regulation of AKT, identifying a crosstalk mechanism involving CXCR4/YY1. Thus, YY1 may represent a crucial molecular target for antiangiogenic therapy during cancer progression.

The field of nitric oxide (NO) research has developed in explosive proportions since the discovery of endogenous NO in 1986. The biological importance of NO was first shown by the findings that nitroglycerin causes vasodilation by liberating NO in the smooth muscle, and activating guanylate cyclase to raise smooth muscle levels of cyclic GMP. NO also inhibits platelet aggregation by cyclic GMP mechanisms. NO activates guanylate cyclase by heme dependent mechanisms involving the formation of a nitrosyl‐heme complex. The high pharmacological potency of NO was finally understood when NO was shown to be formed endogenously, and to be the same as EDRF. Based on these properties of NO, new drugs can be developed as vasodilators and antiplatelet agents for the treatment of a variety of vascular disorders including impotency. NO elicits many other actions in mammalian systems including inhibition of cell proliferation, airway bronchodilation, antimicrobial effects, other host defense effects, and also modulates learning and memory as well as other central functions. This allows for an extensive opportunity to develop novel drugs for the diagnosis, prevention, and treatment of a number of different diseases, many of which are vascular in origin.

Hydrogen sulfide (H ₂ S) is synthesized by 2 enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). l -Cysteine ( l -Cys) acts as a natural substrate for the synthesis of H ₂ S. Human penile tissue possesses both CBS and CSE, and tissue homogenates efficiently convert l -Cys to H ₂ S. CBS and CSE are localized in the muscular trabeculae and the smooth-muscle component of the penile artery, whereas CSE but not CBS is also expressed in peripheral nerves. Exogenous H ₂ S [sodium hydrogen sulfide (NaHS)] or l -Cys causes a concentration-dependent relaxation of strips of human corpus cavernosum. l -Cys relaxation is inhibited by the CBS inhibitor, aminoxyacetic acid (AOAA). Electrical field stimulation of human penile tissue, under resting conditions, causes an increase in tension that is significantly potentiated by either propargylglycine (PAG; CSE inhibitor) or AOAA. In rats, NaHS and l -Cys promote penile erection, and the response to l -Cys is blocked by PAG. Our data demonstrate that the l -Cys/H ₂ S pathway mediates human corpus cavernosum smooth-muscle relaxation.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae . Nitric oxide (NO) and its precursor l -arginine ( l -arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l -arg (1–30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l -arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella -infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l -arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

Nebivolol is a racemic combination of d‐nebivolol (+SRRR nebivolol) and l‐nebivolol (–RSSS nebivolol) that differs chemically from other β‐blockers, with an absolutely symmetrical configuration developing from a central nitrogen atom. D‐nebivolol and l‐nebivolol divaricate pharmacologically and therapeutically, with a noticeably different profile from that of conventional β‐blockers; for instance, the selective blocking of β₁‐adrenoceptors is determined almost exclusively by d‐nebivolol. Both enantiomers act synergistically with respect to blood pressure reduction: the effect of nebivolol on heart rate is exclusively exerted by d‐nebivolol, with these hypotensive effects enhanced by the addition of the l‐enantiomer, which in itself does not influence systolic and diastolic blood pressure. Furthermore, this pronounced and lasting blood pressure reduction is roughly equal to the effect of conventional β‐blockers in high doses. In certain vascular districts, nebivolol stimulates endothelial nitric oxide (NO) synthesis, thereby increasing the availability of NO in the endothelium, smooth muscle, and platelets and, consequently, producing a sustained vasodilation, with decreases in peripheral resistance and blood pressure. These effects are not shared by other β‐adrenoceptor blockers used as references and mainly rely on the l‐enantiomer. L‐nebivolol also increases NO availability under conditions of oxidative stress by the inhibition of endothelial NO synthase (eNOS) uncoupling, thereby reducing NO inactivation. Furthermore, neither nebivolol nor its enantiomers show any intrinsic sympathomimetic activity and undesirable β‐blocker effects, such as a decrease in cardiac output, which do not occur or are less pronounced with the combination of d‐nebivolol and l‐nebivolol. In conclusion, the independent pharmacologic and clinical effects of d‐nebivolol and l‐nebivolol act synergistically to produce a cardiovascular profile that differs noticeably from that of conventional β‐blockers.

Ornithine decarboxylase (ODC) is the first and rate-controlling enzyme in the synthesis of polyamines, which are essential for normal cell growth. We have previously demonstrated that IL-4 and IL-13 can stimulate rat aortic smooth muscle cell (RASMC) proliferation. The objective of this study was to determine whether IL-4 and IL-13 induce cell proliferation by upregulating ODC expression in RASMC. The results revealed that incubation of RASMC with IL-4 and IL-13 for 24 h caused four- to fivefold induction of ODC catalytic activity. The increased ODC catalytic activity was attributed to the increased expression of ODC mRNA. Moreover, these observations were paralleled by increased production of polyamines. We further investigated the signal transduction pathways responsible for ODC induction by IL-4 and IL-13. The data illustrated that PD-98059, a MEK (MAPK kinase) inhibitor, LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and H-89, a protein kinase A (PKA) inhibitor, substantially decreased the induction of ODC catalytic activity and ODC mRNA expression induced by IL-4 and IL-13, suggesting positive regulation of the ODC gene by ERK, PI3K, and PKA pathways. Interestingly, dexamethasone, a known inhibitor of cell proliferation, completely abrogated the response of RASMC to IL-4 and IL-13. Furthermore, the inhibition of ODC by these inhibitors led to the reduced production of polyamines and decreased DNA synthesis as monitored by [³H]thymidine incorporation. Our data indicate that upregulation of ODC by IL-4 and IL-13 might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.

Background: Ovarian carcinoma is the leading cause of mortality among gynecological cancers in the world. The high mortality rate is associated with lack of early diagnosis and development of drug resistance. The antitumor efficacy and mechanism of NCX-4040, a nitric oxide-releasing aspirin derivative, against ovarian cancer is studied. Methods: NCX-4040, alone or in combination with cisplatin (cis-diamminedichloroplatinum, cDDP), was studied in cisplatin-sensitive (A2780 WT) and cisplatin-resistant (A2780 cDDP) cell lines as well as xenograft tumors grown in nude mice. Electron paramagnetic resonance (EPR) was used for measurements of nitric oxide and redox state. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice was used for mechanistic studies. Results: Cells treated with NCX-4040 (25 μM) showed a significant reduction of cell viability (A2780 WT, 34.9 ± 8.7%; A2780 cDDP, 41.7 ± 7.6%; p < 0.05). Further, NCX-4040 significantly enhanced the sensitivity of A2780 cDDP cells (cisplatin alone, 80.6 ± 11.8% versus NCX-4040+cisplatin, 26.4 ± 7.6%; p < 0.01) and xenograft tumors (cisplatin alone, 74.0 ± 4.4% versus NCX-4040+cisplatin, 56.4 ± 7.8%; p < 0.05), to cisplatin treatment. EPR imaging of tissue redox and thiol measurements showed a 5.5-fold reduction (p < 0.01) of glutathione in NCX-4040-treated A2780 cDDP tumors when compared to untreated controls. Immunoblotting analysis of A2780 cDDP tumor xenografts from mice treated with NCX-4040 and cisplatin revealed significant downregulation of pEGFR (Tyr845 and Tyr992) and pSTAT3 (Tyr705 and Ser727) expression. Conclusion: The results suggested that NCX-4040 could resensitize drug-resistant ovarian cancer cells to cisplatin possibly by depletion of cellular thiols. Thus NCX-4040 appears to be a potential therapeutic agent for the treatment of human ovarian carcinoma and cisplatin-resistant malignancies.