Blockade Of COX-2 Research Articles

Activation of Nrf2 in Mice Causes Early Microvascular Cyclooxygenase-Dependent Oxidative Stress and Enhanced Contractility.

Nuclear factor erythroid factor E2-related factor 2 (Nrf2) transcribes antioxidant genes that reduce the blood pressure (BP), yet its activation with tert-butylhydroquinone (tBHQ) in mice infused with angiotensin II (Ang II) increased mean arterial pressure (MAP) over the first 4 days of the infusion. Since tBHQ enhanced cyclooxygenase (COX) 2 expression in vascular smooth muscle cells (VSMCs), we tested the hypothesis that tBHQ administration during an ongoing Ang II infusion causes an early increase in microvascular COX-dependent reactive oxygen species (ROS) and contractility. Mesenteric microarteriolar contractility was assessed on a myograph, and ROS by RatioMaster™. Three days of oral tBHQ administration during the infusion of Ang II increased the mesenteric microarteriolar mRNA for p47phox, the endothelin type A receptor and thromboxane A2 synthase, and increased the excretion of 8-isoprostane F2α and the microarteriolar ROS and contractions to a thromboxane A2 (TxA2) agonist (U-46,619) and endothelin 1 (ET1). These were all prevented in Nrf2 knockout mice. Moreover, the increases in ROS and contractility were prevented in COX1 knockout mice with blockade of COX2 and by blockade of thromboxane prostanoid receptors (TPRs). In conclusion, the activation of Nrf2 over 3 days of Ang II infusion enhances microarteriolar ROS and contractility, which are dependent on COX1, COX2 and TPRs. Therefore, the blockade of these pathways may diminish the early adverse cardiovascular disease events that have been recorded during the initiation of Nrf2 therapy.

Ameliorative effect of fluvoxamine against colon carcinogenesis via COX-2 blockade with oxidative and metabolic stress reduction at the cellular, molecular and metabolic levels

Fluvoxamine's (FLX's) anticancer potential was investigated in pre-clinical research utilizing a DMH-induced colorectal cancer (CRC) rat model. qRT-PCR and immunoblotting validated the mechanistic investigation. The CRC condition was induced in response to COX-2 and IL-6, however, following FLX therapy, the condition returned to normal. FLX's anti-CRC potential may be attributable to COX-2 inhibition since this molecular activity was more apparent for COX-2 than IL-6. FLX repaired the altered metabolites linked to CRC rats, according to 1H-NMR analysis. FLX was shown to be similar to 5-FU in terms of tumor protection, which may be useful in future medication development.

Endothelial prostaglandin D2 opposes angiotensin II contractions in mouse isolated perfused intracerebral microarterioles.

Hypothesis:A lack of contraction of cerebral microarterioles to Ang II (“resilience”) depends on cyclooxygenase (COX) and lipocalin type prostaglandin D sythase L-PGDS producing PGD2 that activates prostaglandin D type 1 receptors (DP1Rs) and nitric oxide synthase (NOS).Materials & Methods:Contractions were assessed in isolated, perfused vessels and NO by fluorescence microscopy.Results:The mRNAs of penetrating intraparenchymal cerebral microarterioles versus renal afferent arterioles were >3000-fold greater for L-PGDS and DP1R and 5-fold for NOS III and COX 2. Larger cerebral arteries contracted with Ang II. However, cerebral microarterioles were entirely unresponsive but contracted with endothelin 1 and perfusion pressure. Ang II contractions were evoked in cerebral microarterioles from COX1 –/– mice or after blockade of COX2, L-PGDS or NOS and in deendothelialized vessels but effects of deendothelialization were lost during COX blockade. NO generation with Ang II depended on COX and also was increased by DP1R activation.Conclusion:The resilience of cerebral arterioles to Ang II contractions is specific for intraparenchymal microarterioles and depends on endothelial COX1 and two products that are metabolized by L-PGDS to generate PGD2 that signals via DP1Rs and NO.

Oxidized-LDL inhibits testosterone biosynthesis by affecting mitochondrial function and the p38 MAPK/COX-2 signaling pathway in Leydig cells

Abnormal lipid/lipoprotein metabolism induced by obesity may affect spermatogenesis by inhibiting testosterone synthesis in Leydig cells. It is crucial to determine which components of lipoproteins inhibit testosterone synthesis. Circulating oxidized low-density lipoprotein (oxLDL), the oxidized form of LDL, has been reported to be an independent risk factor for decreased serum testosterone levels. However, whether oxLDL has a damaging effect on Leydig cell function and the detailed mechanisms have been rarely studied. This study first showed the specific localization of oxLDL and mitochondrial structural damage in testicular Leydig cells of high-fat diet-fed mice in vivo. We also found that oxLDL reduced the mitochondrial membrane potential (MMP) by disrupting electron transport chain and inhibited testosterone synthesis-related proteins and enzymes (StAR, P450scc, and 3β‑HSD), which ultimately led to mitochondrial dysfunction and decreased testosterone synthesis in Leydig cells. Further experiments demonstrated that oxLDL promoted lipid uptake and mitochondrial dysfunction by inducing CD36 transcription. Meanwhile, oxLDL facilitated COX2 expression through the p38 MAPK signaling pathway in Leydig cells. Blockade of COX-2 attenuated the oxLDL-induced decrease in StAR and P450scc. Our clinical results clarified that the increased serum oxLDL level was associated with a decline in circulating testosterone levels. Our findings amplify the damaging effects of oxLDL and provide the first evidence that oxLDL is a novel metabolic biomarker of male-acquired hypogonadism caused by abnormal lipid metabolism.

Commensal dysbiosis modulates the tumor microenvironment in breast cancer and diminishes efficacy of PD-L1 inhibition

Abstract For patients with HR+ breast cancer, non-responsiveness to PD-1/PD-L1 blockade cannot be attributed to poor immunogenicity, as immune-stimulatory gene signatures are associated with greater long-term survival and enhanced responsiveness to therapy. Targeting myeloid-driven immune suppression has also been shown to enhance efficacy of PD-L1 blockade in non-responsive tumors. However, we find that in HR+ breast cancer, synergy with PD-L1 blockade is negatively influenced by a pre-established loss of commensal homeostasis, or commensal dysbiosis. Using a clinically relevant non-responsive HR+ breast tumor model, establishing commensal dysbiosis prior to breast tumor initiation drives significant and irreversible T cell dysfunction within primary tumors. Additionally, commensal dysbiosis alters the cellular composition within the tumor microenvironment, culminating in the recruitment and polarization of M2-like macrophages with distinct tumor-suppressive attributes. Systemically, tumor-bearing animals with established commensal dysbiosis have significantly higher levels of prostaglandin E2 with the serum, which is independent of tumor volume. However, blockade of COX-2 only synergizes with PD-L1 blockade in animals without commensal dysbiosis. Unexpectedly, we found that commensal dysbiosis enhances COX-1/PGE2 signaling within the mammary epithelium during early tumor progression. Inhibition of COX-1-mediated PGE2 synthesis during early stages of tumor progression resulted in delayed tumor progression only in dysbiotic tumor-bearing mice, suggesting that dysbiosis-induced COX-1 mediated tissue inflammation is sufficient to drive irreversible immune suppression during advanced breast cancer.

Novel 1,4-benzothazines obliterate COX-2 mediated JAK-2/STAT-3 signals with potential regulation of oxidative and metabolic stress during colorectal cancer

1,4-benzothiazines have ameliorative effects through inhibition of COX-2 mediated STAT-3 pathways at G-protein couple receptor site. As per this scenario, we recently prepared and tested novel 1,4-benzothiazine derivatives against HT-29 human colon cancer cell line. Two compounds namely AR13 and AR15 showed higher inhibitions among all the synthesized compounds. In the present context, we conducted the in vivo antiproliferative action and identified the molecular mechanism associated to cytotoxic action of AR13 and AR15 in dimethylhydrazine (DMH) induced colorectal carcinoma (CRC) model. Various physiological, oxidative stress, histopathology, ELISA, qRT-PCR, western blot and NMR-based metabolomics were accomplished to evaluate the anticancer effect of titled compounds. Both compounds were subjected to histological and biochemical tests to observe the protective action of the compounds. ELISA showed potential role of these compounds to normalize increased levels of IL-2, IL-6 and COX-2 mediators. This action was more pronounced for COX-2 rather than IL-2 and IL-6. Gene expression analyses further revealed that both of them attenuated the over-expressed COX-2 gene. Furthermore, it was confirmed that these compounds exerted antitumor potential via preventing COX-2 induced JAK-2 and STAT-3 phosphorylation. This action was substansiated by immunohistochemistry using JAK2, p-JAK2, STAT3 and p-STAT3 targets in colon tissue. Finally, score plots of PLS-DA models exhibited significant metabolic discriminations between the treated and CRC groups, and both compounds showed ability to restore the imbalance of multiple metabolites during CRC. In conclusion, our study provided the evidence towards better antiproliferative effect of AR13 and AR15 in DMH-induced CRC through the blockade of COX-2/JAK-2/STAT-3 signal transduction pathway and could be demonstrated as useful anti-CRC candidate molecules for future anticancer therapy.

Role of ALDH1 in the prognosis of esophageal cancer and its relationship with tumor microenvironment.

Aldehyde dehydrogenase 1 (ALDH1) is associated with tumorigenesis, and significantly increased in cancer stem-like cells. In the present study, the role of ALDH1 in esophageal squamous cell carcinoma (ESCC) was investigated. We retrospectively analyzed the clinical outcomes of 148 ESCC and examined its correlation with ALDH1 levels. Furthermore, we preformed cellular and animal experiments to investigate the role of ALDH1 in tumor progression and microenvironment. Our data revealed that ALDH1 staining was positively linked to a higher clinical stage, higher loco-regional failure rate, and shorter survival time. Furthermore, there was a positive link between ALDH1 expression and IL-6 signaling according to the data of clinical specimens and cellular experiments. Using animal model, ALDH1-positive tumors were associated with aggressive tumor growth, increased IL-6, augmented epithelial-mesenchymal transition (EMT), and activation of myeloid-derived suppressor cells (MDSCs). Blockade of COX-2 attenuated the aggressive tumor growth of ALDH1-positive cancer cells. In conclusion, our findings suggested that ALDH1 played an important role in tumor aggressiveness and associated with a tumor-promoting microenvironment in esophageal cancer. Directly targeting ALDH1 or using a COX-2 inhibitor could be a promising strategy for the treatment of ESCC.

Abstract P344: Nuclear Factor E2-related Factor 2 (nrf2) Causes Early Microvascular Endothelial Dysfunction and Increased Adma by Activation of the Cox2/tp Receptor Pathway in Mice Infused With Angiotensin II for Three Days

Background: Activation of Nrf2 by tert-buthylhydroquinone (tBHQ) prevents hypertension, oxidative stress, endothelial dysfunction and ADMA in mice infused with angiotensin II (Ang II) for 12 days. However, Nrf2 activation with bardoxolone methyl increased cardiovascular events in diabetic patients within one week. Since we found that tBHQ activated cyclooxygenase (COX)-2 within 3 days, we tested the hypothesis that short-term tBHQ administration to Ang II infused mice increases microvascular dysfunction via COX dependent of PGs and TxA 2 that activate thromboxane-prostanoid receptors (TP-Rs). Methods: Mesenteric resistance arterioles(MRAs)were isolated from mice infused for 3 days with ANG II (400 ng/kg/min) or vehicle and given oral tBHQ (0.1% of water) or vehicle (n=6 mice/group). Endothelial derived relaxation factor (EDRF) was assessed by a myograph and NO and ROS by RatioMaster TM . Results: Compared to vehicle, Ang II infused mice given tBHQ had increased (P<0.05) conscious mean arterial pressure (135±6 vs 115±7 mmHg), urinary 8-Iso prostane (1.5±0.4 vs 1.1±0.2ng/mg creatinine), and decreased EDRF (17± 3 vs 23± 2%) and NO (0.23 ± 0.02 vs 0.35 ± 0.02 Δunits), and enhanced ( P<0.05, ) cellular ROS (0.27 ± 0.02 vs 0.12 ± 0.03 Δunits), mitochondria ROS (0.24 ± 0.02 vs 0.1 ± 0.02 Δunit) and microvascular asymmetric dimethylarginine (ADMA, 77 ± 6 vs 55 ± 7 nmol/mg protein). All these effects of tBHQ were prevented in COX-1 knockout mice drinking parecoxib (COX-2 inhibitor) and in TPR and Nrf2 knockout mice. Conclusions: Activation of Nrf2 increases short term Ang II-induced increases in BP, oxidative stress, ADMA and endothelial dysfunction. These depend on signaling via COX 1 + 2 and TP receptors. Our studies reveal novel dual effects of Nrf2 in Ang II infused mice: increased BP, ROS, ADMA and endothelial dysfunction within 3 days secondary to activation of COX/TPR signaling follow by the opposite effects of Nrf2 on BP, ADMA and vascular function within 12 days. Therefore, blockade of COX-2/TPRs pathway during initiation of Nrf2 therapy may prevent the devastating early adverse effects that have prevented its use in hypertension or diabetes.

Inhibition of LPS induced pro-inflammatory responses in RAW 264.7 macrophage cells by PVP-coated naringenin nanoparticle via down regulation of NF-κB/P38MAPK mediated stress signaling.

Naringenin (NAR) was found to display strong pharmacological properties. Since the clinical relevance of NAR is limited by its low bioavailability, we effectively synthesized and characterized a novel PVP-coated NAR nanoparticle (NAR NP) to enhance the therapeutic efficacy of NAR. The present study was designed to investigate the effects of NAR NP on lipopolysaccharide (LPS) induced inflammatory response in RAW 264.7 macrophage cells. In vitro cell culture studies of LPS stimulated RAW 264.7 macrophage cells were used as experimental model. Cytotoxicity studies revealed that NAR NP is safe even at maximum tested concentration of 200μg/ml. Initial dose fixation study in LPS induced RAW 264.7 cells, revealed the minimum optimal concentration required for anti inflammatory effect as 25μg/ml. mRNA expression studies showed that NAR NP significantly down regulated the expressions of NF-κB and P38MAPK, which is paralleled with the inhibition of the nuclear translocation of NF-κB. This in turn led to the blockade of iNOS and COX-2, thereby inhibiting the production of nitric oxide and pro inflammatory cytokines such as TNF-α, IL-6, MCP-1 and IL-1β. NAR NP was found to be more efficient, when compared with NAR. Anti-inflammatory effects of NAR NP may be allocated to the down-regulation of COX -2 and iNOS via the suppression of NF-κB and MAPK signaling pathway in RAW 264.7 macrophages. The data suggests that NAR NP can be used as a potent candidate for the treatments of inflammatory diseases by exploiting the nanoscale properties and targeting efficacy.

Effects of pro-inflammatory cytokines, lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE2, PGF2α) or their corresponding ethanolamides (PGE2-EA or PGF2α-EA) over 48h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10−5M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10−4M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE2 transiently increased Caco-2 monolayer permeability at 24h, while LPS (10μg/ml) increased permeability over 24–48h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE2 transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.

Hydrogen sulfide promotes cell proliferation of oral cancer through activation of the COX2/AKT/ERK1/2 axis.

Hydrogen sulfide, the third gaseous transmitter, is one of the main causes of halitosis in the oral cavity. It is generally considered as playing a deleterious role in many oral diseases including oral cancer. However, the regulatory mechanisms involved in the effects of hydrogen sulfide on oral cancer growth remain largely unknown. In the present study, we investigated the underlying mechanisms through CCK-8 assay, EdU incorporation, real-time PCR, western blot and pathway blockade assays. Our results showed that hydrogen sulfide promoted oral cancer cell proliferation through activation of the COX2, AKT and ERK1/2 pathways in a dose-dependent manner. Blocking any of the three above pathways inhibited hydrogen sulfide-induced oral cancer cell proliferation. Meanwhile, blockade of COX2 by niflumic acid downregulated NaHS-induced p-ERK and p-AKT expression. Inactivation of the AKT pathway by GSK690693 significantly decreased NaHS‑induced p-ERK1/2 expression, and inhibition of the ERK1/2 pathway by U0126 markedly increased NaHS-induced p-AKT expression. Either the AKT or ERK1/2 inhibitor did not significantly alter the COX2 expression level. Our data revealed, for the first time, that hydrogen sulfide promotes oral cancer cell proliferation through activation of the COX2/AKT/ERK1/2 axis, suggesting new potential targets to eliminate the effect of hydrogen sulfide on the development of oral cancer.

The Combined Blockade of β-Adrenoceptor and COX-2 During the Perioperative Period to Improve Long-term Cancer Outcomes.

The Combined Blockade of β-Adrenoceptor and COX-2 During the Perioperative Period to Improve Long-term Cancer Outcomes.

A primary role for kinin B1 receptor in inflammation, organ damage, and lethal thrombosis in a rat model of septic shock in diabetes.

Diabetes mellitus and septic shock increase the incidence of mortality by thrombosis. Although kinin B1 receptor (B1R) is involved in both pathologies, its role in platelet function and thrombosis remains unknown. This study investigates the expression, the inflammatory, and pro-thrombotic effects of B1R in a model of septic shock in diabetic rats. Sprague-Dawley rats were made diabetic with streptozotocin (STZ) (65 mg/kg, i.p.). Four days later, control and STZ-diabetic rats were injected with lipopolysaccharide (LPS) (2 mg/kg, i.p.) or the vehicle. B1R antagonist (SSR240612, 10 mg/kg by gavage) was given either acutely (12 and 24 h prior to endpoint analysis) or daily for up to 7 days. Moreover, a 7-day treatment was given either with cyclooxygenase (COX)-2 inhibitor (niflumic acid, 5 mg/kg, i.p.), non-selective COX-1 and COX-2 inhibitor (indomethacin, 10 mg/kg, i.p.), non-selective nitric oxide synthase (NOS) inhibitor (L-NAME, 50 mg/kg by gavage), iNOS inhibitor (1400W, 5 mg/kg, i.p.), or heparin (100 IU/kg, s.c.). The following endpoints were measured: edema and vascular permeability (Evans blue dye), B1R expression (qRT-PCR, western blot, flow cytometry), aggregation in platelet-rich plasma (optical aggregometry), and organ damage (histology). Rats treated with STZ, LPS, and STZ plus LPS showed significant increases in edema and vascular permeability (heart, kidney, lung, and liver) and increased expression of B1R in heart and kidney (mRNA) and platelets (protein). Lethal septic shock induced by LPS was enhanced in STZ-diabetic rats and was associated with lung and kidney damage, including platelet micro-aggregate formation. SSR240612 prevented all these abnormalities as well as STZ-induced hyperglycemia and LPS-induced hyperthermia. Similarly to SSR240612, blockade of iNOS and COX-2 improved survival. Data provide the first evidence that kinin B1R plays a primary role in lethal thrombosis in a rat model of septic shock in diabetes. Pharmacological rescue was made possible with B1R antagonism or by inhibition of iNOS and COX-2, which may act as downstream mechanisms.

Cyclooxygenase-2 blockade can improve efficacy of VEGF-targeting drugs.

Anti-angiogenic therapies were approved for different cancers. However, significant primary and secondary resistance hampers efficacy in several tumor types including breast cancer. Thus, we need to develop clinically applicable strategies to enhance efficacy of anti-angiogenic drugs.We report that anti-angiogenic therapies can induce upregulation of cyclooxygenase-2 (Cox-2) and of its product prostaglandin E2 (PGE2) in breast cancer models. Upon Cox-2 inhibition PGE2 levels were normalized and efficacy of anti-vascular endothelial growth factor receptor 2 (anti-VEGFR-2) antibodies and sunitinib was enhanced. Interestingly, both treatments exerted additive anti-angiogenic effects. Following Cox-2 inhibition, we observed reduced infiltration of tumors with cancer-associated fibroblasts (CAFs) and lower levels of pro-angiogenic factors active besides the VEGF axis including hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2). Mechanistic studies indicated that Cox-2 inhibition reduced PGE2-induced migration and proliferation of CAFs via inhibiting phosphorylation of Akt.Hence, Cox-2 inhibition can increase efficacy of anti-angiogenic treatments and our findings might pave the road for clinical investigations of concomitant blockade of Cox-2 and VEGF-signaling.

Prenatal nicotine exposure enhances Cx43 and Panx1 unopposed channel activity in brain cells of adult offspring mice fed a high-fat/cholesterol diet.

Nicotine, the most important neuroteratogen of tobacco smoke, can reproduce brain and cognitive disturbances per se when administered prenatally. However, it is still unknown if paracrine signaling among brain cells participates in prenatal nicotine-induced brain impairment of adult offspring. Paracrine signaling is partly mediated by unopposed channels formed by connexins hemichannels (HCs) and pannexins serving as aqueous pores permeable to ions and small signaling molecules, allowing exchange between the intra- and extracellular milieus. Our aim was to address whether prenatal nicotine exposure changes the activity of those channels in adult mice offspring under control conditions or subjected to a second challenge during young ages: high-fat/cholesterol (HFC) diet. To induce prenatal exposure to nicotine, osmotic minipumps were implanted in CF1 pregnant mice at gestational day 5 to deliver nicotine bitartrate or saline (control) solutions. After weaning, offspring of nicotine-treated or untreated pregnant mice were fed ad libitum with chow or HFC diets for 8 weeks. The functional state of connexin 43 (Cx43) and pannexin 1 (Panx1) unopposed channels was evaluated by dye uptake experiments in hippocampal slices from 11-week-old mice. We found that prenatal nicotine increased the opening of Cx43 HCs in astrocytes, and Panx1 channels in microglia and neurons only if offspring mice were fed with HFC diet. Blockade of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2) and prostaglandin E receptor 1 (EP1), ionotropic ATP receptor type 7 (P2X7) and NMDA receptors, showed differential inhibition of prenatal nicotine-induced channel opening in glial cells and neurons. Importantly, inhibition of the above mentioned enzymes and receptors, or blockade of Cx43 and Panx1 unopposed channels greatly reduced adenosine triphosphate (ATP) and glutamate release from hippocampal slices of prenatally nicotine-exposed offspring. We propose that unregulated gliotransmitter release through Cx43 and Panx1 unopposed channels may participate in brain alterations observed in offspring of mothers exposed to tobacco smoke during pregnancy.

Abstract 1161: COX-2 blockade improves efficacy of VEGF-targeting drugs

Abstract Efficacy of anti-angiogenic drugs is hampered by hypoxia-induced resistance. Because cyclooxygenase-2 (Cox-2) is upregulated in hypoxic conditions we analyzed mRNA expression levels of cyclooxygenase-1 (Cox-1) and Cox-2 in GFP+ FACS-sorted tumor cells from 4T1 tumors after treatment with anti-VEGFR2 antibodies (DC101) or with sunitinib. Cox-2 but not Cox-1 mRNA was upregulated by 2.3-fold upon anti-angiogenic treatment. In addition we found 5.2-fold increased prostaglandin E2 levels in 4T1 tumors after anti-angiogenic therapy. We hypothesized that concomitant blockade of Cox-2 could increase efficacy of anti-angiogenic agents. Therefore we treated 4T1 tumor-bearing mice with sunitinib or DC101 alone and in combination with acetylsalicyclic acid (ASA). We found that single treatment with ASA or angiogenesis inhibitors inhibited tumor growth and that combined inhibition of Cox-2 and VEGF(R) signaling exerted additive therapeutic efficacy (n=5; 1142±84 (ASS); 1148±78 (Sunitnib) vs. 63±5 mg (combination); p<0.0001). Similar effects were achieved upon combining the specific Cox2 inhibitor SC-236 with anti-angiogenic therapy (n=6; 985±147 (SC-326); 1123±61 (Sunitnib) vs. 680±76 mg (combination); p<0.05) and in the 6CCL4 orthotopic breast cancer model (data not shown). We carried out an extensive profiling of the tumor cells and their microenvironment upon concomitant blockade of Cox-2 and VEGF signaling in order to elucidate the underlying mechanism. We found no changes in tumor cell proliferation or upon combined Cox-2 inhibition and anti-angiogenic therapies. Also combined Cox-2 and VEGF-signaling inhibition did not change the quantitative composition of the inflammatory tumor infiltrate. However, upon analyzing polarization of FACS-sorted TAMs we found that mRNA of the M1 markers iNOS, MHCII, IL1β and TNFα were upregulated upon treatment with ASA alone and/or in combination with anti-angiogenic agents compared to controls or monotherapy with anti-angiogenic agents. In contrast, the M2 markers Arg1 and YM1 were downregulated upon treatment with Cox2-inhibitors and anti-angiogenic agents. Therefore, Cox-2 inhibitors skew TAMs towards an anti-tumoral M1 phenotype while the pro-angiogenic, tumor promoting M2-phenotype is suppressed. In addition, Cox-2 and PGE2 can promote tumor angiogenesis. This alternative pro-angiogenic pathway would be enhanced by increased Cox-2 expression and PGE2 levels and could contribute to resistance against anti-angiogenic treatments. In line with this hypothesis the MVD was decreased 4T1 tumors treated with combined Cox-2 and VEGF blockade compared to the respective monotherapy (n=7; 31±2.4 (Sunitinib); 28.58±0.83 (ASS) vs. 9.67±1.71 (combination); p<0.001). In conclusion concomitant Cox2 inhibition and anti-angiogenic therapies exert pronounced additive effects, which are at least partially due to increased M1 polarization and additive anti-angiogenic effects. Citation Format: Isabel Ben Batalla, Miguel Cubas-Cordova, Florian Udonta, Mark Wroblewski, Stefanie Sawall, Victoria Gensch, Klaus Pantel, Carsten Bokemeyer, Sonja Loges. COX-2 blockade improves efficacy of VEGF-targeting drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1161. doi:10.1158/1538-7445.AM2014-1161

Effects of age and sex on cerebrovascular function in the rat middle cerebral artery.

BackgroundAlthough the mechanisms underlying the beneficial effects of estrogen on cerebrovascular function are well known, the age-dependent deleterious effects of estrogen are largely unstudied. It was hypothesized that age and sex interact in modulating cerebrovascular reactivity to vasopressin (VP) by altering the role of prostanoids in vascular function.MethodsFemale (F) Sprague–Dawley rats approximating key stages of “hormonal aging” in humans were studied: premenopausal (mature multigravid, MA, cyclic, 5–6 months) and postmenopausal (reproductively senescent, RS, acyclic, 10–12 months). Age-matched male (M) rats were also studied. Reactivity to VP (10−12–10−7 M) was measured in pressurized middle cerebral artery segments in the absence or presence of selective inhibitors of COX-1 (SC560, SC, 1 μM) or COX-2 (NS398, NS, 10 μM). VP-stimulated release of PGI2 and TXA2 were measured using radioimmunoassay of 6-keto-PGF1α and TXB2 (stable metabolites, pg/mg dry wt/45 min).ResultsIn M, there were no changes in VP-induced vasoconstriction with age. Further, there were no significant differences in basal or in low- or high-VP-stimulated PGI2 or TXA2 production in younger or older M. In contrast, there were marked differences in cerebrovascular reactivity and prostanoid release with advancing age in F. Older RS F exhibited reduced maximal constrictor responses to VP, which can be attributed to enhanced COX-1 derived dilator prostanoids. VP-induced vasoconstriction in younger MA F utilized both COX-1 and COX-2 derived constrictor prostanoids. Further, VP-stimulated PGI2 and TXA2 production was enhanced by endogenous estrogen and decreased with advancing age in F, but not in M rats.ConclusionsThis is the first study to examine the effects of age and sex on the mechanisms underlying cerebrovascular reactivity to VP. Interestingly, VP-mediated constriction was reduced by age in F, but was unchanged in M rats. Additionally, it was observed that selective blockade of COX-1 or COX-2 produced age-dependent changes in cerebrovascular reactivity to VP and that VP-stimulated PGI2 and TXA2 production were enhanced by endogenous estrogen in younger F. A better understanding of the mechanisms by which estrogen exerts its effects may lead to new age- and sex-specific therapeutic agents for the prevention and/or treatment of cerebrovascular diseases.

Abstract 066: Cyclooxygenase-depended Signaling Via Thromboxane Prostanoid Receptors Mediates Endothelin-i Induced Ros Generation In Mesenteric Resistance Vessels From Mice Infused With Angiotensin Ii

Background: Angiotensin II (Ang II) increases reactive oxygen species (ROS) and contractions to thromboxane and endothelin-1 (ET-1). Therefore, we tested the hypothesis that cyclooxygenase (COX) and/or thromboxane-prostanoid receptors (TP-Rs) mediate enhanced ROS generations with ET-1 in Ang II-infused mice. Methods: ROS was assessed by urinary 8-isoprotane(8-Iso) excretion and ethedium : dihydroetheldium (DHE) in mesenteric resistance arterioles (MRAs) from wild type (+/+) and littermate COX-1 -/- or TP-R -/- mice infused with vehicle or angiotensin II (Ang II, 400 ng/kg/min for 14 days) (n=6/ group, mean ±SEM). Results: Ang II infusion increased excretion (ng/mg creatine) of TxB 2 (1.3±0.1±1.0±0.1 in COX-1 +/+ mice and 1.9±0.1 vs 1.2±0.1 in TP-R +/+ mice, all P<0.05) and 8-Iso (2.1±0.2 vs 1.4±0.1 in COX-1 +/+ mice and 2.2±0.1 vs 1.4±0.2 in TP-R +/+ mice, all P<0.05) but not in COX-1 -/- or TP-R -/- mice. Ang II enhanced ROS generation (Δunit) with 10 -7 M ET-1 in MRAs from both +/+ mouse genotypes (1.7±0.2 vs 0.1±0.1 in COX-1 +/+ mice and 3.2±0.3 vs 0.1±0.1 in TP-R +/+ mice, all P<0.01). However, this increase in ROS was fully prevented in TP-R-/- mouse vessels (0.3±0.2 vs 0.2±0.1, NS) and in COX-1 +/+ mouse vessels after combined blockade of COX-1( 10 -5 M SC-560) and -2 (paracoxib 10 -5 M) (0.2±0.1 vs 0.1±0.1, NS) and in COX-1 -/- mouse vessels after paracoxib (0.2±0.1 vs 0.2±0.2, NS). Increased ROS generation was only partially prevented in COX-1 -/- mouse vessels (0.5±0.1 vs 0.2±0.2, P<0.05) or in COX-1 +/+ mouse vessels after blockade of COX-1 ( 0.7±0.1 vs 0.1±0.1, NS) or COX-2 (1.0±0.1 vs 0.1±0.1,P<0.05). Conclusions: Increased ROS generation with ET-1 in microvessels from Ang II infused mice depends on products of both COX-1 and -2 that activate TP-Rs. Thus, combined blockade of COX-1 and -2 or TP-Rs may prevent vascular ROS and its many complications during increased Ang II and ET-1, such as hypertension, hypoxia or shock.

The Role of Cyclooxygenase-1 and -2 in Sevoflurane-Induced Postconditioning Against Myocardial Infarction

Cyclooxygenase (COX)-2 mediates ischemic pre- and postconditioning as well as anesthetic-induced preconditioning. However, the role of COX-1 and -2 in anesthetic-induced postconditioning has not been investigated. We evaluated the role of COX-1 and -2 in sevoflurane-induced postconditioning in vivo. Pentobarbital-anaesthetized male C57BL/6 mice were subjected to 45 minutes of coronary artery occlusion and 3 hours of reperfusion. Animals received either no intervention, the vehicle dimethyl sulfoxide (DMSO, 10 µL/g intraperitoneally), acetylsalicylic acid (ASA, 5 µg/g intraperitoneally), the selective COX-1 inhibitor SC-560 (10 µg/g intraperitoneally), or the selective COX-2 inhibitor NS-398 (5 µg/g intraperitoneally). 1.0 MAC (minimum alveolar concentration) sevoflurane was administered for 18 minutes during early reperfusion either alone or in combination with ASA, SC-560, and NS-398. Infarct size was determined with triphenyltetrazolium chloride. Statistical analysis was performed using 1-way and 2-way analyses of variance with post hoc Duncan testing. The infarct size in the control group was 44% ± 9%. DMSO (42% ± 7%), ASA (36% ± 6%), and NS-398 (44% ± 18%) had no effect on infarct size. Sevoflurane (17% ± 4%; P < .05) and SC-560 (26% ± 10%; P < .05) significantly reduced the infarct size compared with control condition. Sevoflurane-induced postconditioning was not abolished by ASA (16% ± 5%) and SC-560 (22% ± 4%). NS-398 abolished sevoflurane-induced postconditioning (33% ± 14%). It was concluded that sevoflurane induces postconditioning in mice. Inhibition of COX-1 elicits a myocardial infarct size reduction and does not abolish sevoflurane-induced postconditioning. Blockade of COX-2 abolishes sevoflurane-induced postconditioning. These results indicate that sevoflurane-induced postconditioning is mediated by COX-2.

Inflammation induced by mast cell deficiency rather than the loss of interstitial cells of Cajal causes smooth muscle dysfunction in W/Wv mice

The initial hypothesis suggested that the interstitial cells of Cajal (ICC) played an essential role in mediating enteric neuronal input to smooth muscle cells. Much information for this hypothesis came from studies in W/Wv mice lacking ICC. However, mast cells, which play critical roles in regulating inflammation in their microenvironment, are also absent in W/Wv mice. We tested the hypothesis that the depletion of mast cells in W/Wv mice generates inflammation in fundus muscularis externa (ME) that impairs smooth muscle reactivity to Ach, independent of the depletion of ICC. We performed experiments on the fundus ME from wild type (WT) and W/Wv mice before and after reconstitution of mast cells by bone marrow transplant. We found that mast cell deficiency in W/Wv mice significantly increased COX-2 and iNOS expression and decreased smooth muscle reactivity to Ach. Mast cell reconstitution or concurrent blockade of COX-2 and iNOS restored smooth muscle contractility without affecting the suppression of c-kit in W/Wv mice. The expression of nNOS and ChAT were suppressed in W/Wv mice; mast cell reconstitution did not restore them. We conclude that innate inflammation induced by mast cell deficiency in W/Wv mice impairs smooth muscle contractility independent of ICC deficiency. The impairment of smooth muscle contractility and the suppression of the enzymes regulating the synthesis of Ach and NO in W/Wv mice need to be considered in evaluating the role of ICC in regulating smooth muscle and enteric neuronal function in W/Wv mice.