Abstract 44: VEGF Inhibitor-induced Vascular Dysfunction Is Ameliorated By PARP/TRPM2 Inhibition

Hypertension is a common unwanted effect of VEGF inhibitors (VEGFi), which are used as anti-angiogenic drugs in cancer treatment. Clinical observations suggest that the combination of VEGFi with another anti-cancer drug, olaparib (PARP inhibitor [PARPi]), may attenuate the development of hypertension. However putative vascular mechanisms are unknown. PARP plays a major role in the activation of TRPM2, a redox-sensitive Ca 2+ channel, which is associated with hypertension-induced vascular dysfunction. We hypothesized that PARPi attenuates VEGFi-induced vascular injury through TRPM2/Ca 2+ -dependent pathways. Human vascular smooth muscle cells (hVSMC), human aortic endothelial cells (HAEC), and mouse mesenteric arteries were studied. Cells/arteries were exposed to axitinib (VEGFi) alone (3μM) or in combination with olaparib (1μM). Wire myography was used to assess vascular function. Axitinib reduced ACh-induced vasodilation (% relaxation: 70.5 [Ct] vs. 34.8 [Axi]), an effect blocked by olaparib. U46619- and ET-1-induced vasoconstriction were increased by axitinib (% KCl- U4 : 101.2 [Ct] vs. 141.4 [Axi]; ET-1 : 122.6 [Ct] vs. 152.5 [Axi]), an effect not observed with axitinib plus olaparib. TRPM2 channel blocker (8-Br-cADPR; 1μM) attenuated the hypercontractile effects and endothelial dysfunction induced by axitinib. Axitinib increased ROS production in hVSMC (RUL: 0.8±0.2 [Ct] vs. 1.1±0.09 [Axi]) and HAEC (0.7±0.4 [Ct] vs. 1.2±0.1 [Axi]), stimulated phosphorylation of the inhibitory site of eNOS (a.u.: 0.99±0.35 [Ct] vs. 1.35±0.10 [Axi]) and induced exaggerated Ca 2+ influx (AUC: 17541±4708 [Ct] vs. 22249±1438 [Axi]) in hVSMC. These effects were blocked by olaparib and 8-Br-cADPR. Axitinib also induced phosphorylation of MLC20 in hVSMC (a.u.: 0.028±0.02 [Ct] vs. 0.04±0.01 [Axi]) and aorta (a.u.: 0.3±0.01 [Ct] vs. 0.5±0.001 [Axi]). Our data indicate that PARP/TRPM2 inhibition attenuates axitinib-mediated vascular dysfunction and normalizes impaired hVSMC and HAEC signalling induced by VEGFi. We define a putative vasoprotective effect of olaparib that may ameliorate vascular injury and hypertension induced by VEGFi in cancer treatment.

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline—a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc—a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole—two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

Deficiency of ROS-Activated TRPM2 Channel Protects Neurons from Cerebral Ischemia-Reperfusion Injury through Upregulating Autophagy

Cerebral ischemia-reperfusion (I-R) transiently increased autophagy by producing excessively reactive oxygen species (ROS); on the other hand, activated autophagy would remove ROS-damaged mitochondria and proteins, which led to cell survival. However, the regulation mechanism of autophagy activity during cerebral I-R is still unclear. In this study, we found that deficiency of the TRPM2 channel which is a ROS sensor significantly decreased I-R-induced neuronal damage. I-R transiently increased autophagy activity both in vitro and in vivo. More importantly, TRPM2 deficiency decreased I-R-induced neurological deficit score and infarct volume. Interestingly, our results indicated that TRPM2 deficiency could further activate AMPK rather than Beclin1 activity, suggesting that TRPM2 inhibits autophagy by regulating the AMPK/mTOR pathway in I-R. In conclusion, our study reveals that ROS-activated TRPM2 inhibits autophagy by downregulating the AMPK/mTOR pathway, which results in neuronal death induced by cerebral I-R, further supporting that TRPM2 might be a potential drug target for cerebral ischemic injury therapy.

Deletion of Mitochondrial Translocator Protein (TSPO) Gene Decreases Oxidative Retinal Pigment Epithelial Cell Death via Modulation of TRPM2 Channel

The current results indicated the possible protective actions of 18 kDa mitochondrial translocator protein (TSPO) deletion on TRPM2 stimulation, mitochondrial free ROS (Mito-fROS) and apoptotic harmful actions in the cells of adult retinal pigment epithelial19 (ARPE19). There was a direct relationship between TSPO and the disease of age-related macular degeneration. The nature of TSPO implicates upregulation of Mito-fROS and apoptosis via the activation of Ca2+ channels in ARPE19, although deletion of TSPO gene downregulates the activation. The decrease of oxidative cytotoxicity and apoptosis might induce in TSPO gene deleted cells by the inhibition of Mito-fROS and PARP-1 activation-induced TRPM2 cation channel activation. The ARPE19 cells were divided into two main groups as TSPO expressing (ARPE19) and non-expressing cells (ARPE19-KO). The levels of caspase -3 (Casp -3), caspase -9 (Casp -9), apoptosis, Mito-fROS, TRPM2 current and intracellular free Ca2+ were upregulated in the ARPE19 by the stimulations of H2O2 and ADP-ribose, although their levels were downregulated in the cells by the modulators of PARP-1 (DPQ and PJ34), TRPM2 (ACA and 2APB) and glutathione. However, the H2O2 and ADP-ribose-mediated increases were not observed in the ARPE19-KO. The expression levels of Bax, Casp -3, Casp -9 and PARP-1 were higher in the ARPE19 group as compared to the ARPE19-KO group. In summary, current results confirmed that TRPM2-mediated cell death and oxidative cytotoxicity in the ARPE19 cells were occurred by the presence of TSPO. The deletion of TSPO may be considered as a therapeutic way to TRPM2 activation-mediated retinal oxidative injury.

Methamphetamine and HIV-Tat Protein Synergistically Induce Oxidative Stress and Blood-Brain Barrier Damage via Transient Receptor Potential Melastatin 2 Channel

Synergistic impairment of the blood-brain barrier (BBB) induced by methamphetamine (METH) and HIV-Tat protein increases the risk of HIV-associated neurocognitive disorders (HAND) in HIV-positive METH abusers. Studies have shown that oxidative stress plays a vital role in METH- and HIV-Tat-induced damage to the BBB but have not clarified the mechanism. This study uses the human brain microvascular endothelial cell line hCMEC/D3 and tree shrews to investigate whether the transient receptor potential melastatin 2 (TRPM2) channel, a cellular effector of the oxidative stress, might regulate synergistic damage to the BBB caused by METH and HIV-Tat. We showed that METH and HIV-Tat damaged the BBB in vitro, producing abnormal cell morphology, increased apoptosis, reduced protein expression of the tight junctions (TJ) including Junctional adhesion molecule A (JAMA) and Occludin, and a junctional associated protein Zonula occludens 1 (ZO1), and increased the flux of sodium fluorescein (NaF) across the hCMEC/D3 cells monolayer. METH and HIV-Tat co-induced the oxidative stress response, reducing catalase (CAT), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) activity, as well as increased reactive oxygen species (ROS) and malonaldehyde (MDA) level. Pretreatment with n-acetylcysteine amide (NACA) alleviated the oxidative stress response and BBB damage characterized by improving cell morphology, viability, apoptosis levels, TJ protein expression levels, and NaF flux. METH and HIV-Tat co-induced the activation and high protein expression of the TRPM2 channel, however, early intervention using 8-Bromoadenosine-5′-O-diphosphoribose (8-Br-ADPR), an inhibitor of TPRM2 channel, or TRPM2 gene knockdown attenuated the BBB damage. Oxidative stress inhibition reduced the activation and high protein expression of the TRPM2 channel in the in vitro model, which in turn reduced the oxidative stress response. Further, 8-Br-ADPR attenuated the effects of METH and HIV-Tat on the BBB in tree shrews—namely, down-regulated TJ protein expression and increased BBB permeability to Evans blue (EB) and NaF. In summary, the TRPM2 channel can regulate METH- and HIV-Tat-induced oxidative stress and BBB injury, giving the channel potential for developing drug interventions to reduce BBB injury and neuropsychiatric symptoms in HIV-infected METH abusers.

Abstract P765: Therapeutic Approaches to Reduce Post-Stroke Cognitive Impairment

Introduction: Cognitive impairments and memory loss are common after stroke, with an emerging awareness of a high risk of conversion to post-stroke dementia. It is increasingly clear that in addition to neuronal injury following cerebral ischemia, impaired functional networks contribute to long-term functional deficits. Synaptic plasticity (LTP) is the leading cellular model of learning and memory. Thus, we utilize electrophysiological recordings of hippocampal LTP as an indicator of network health following ischemia in combination with neurobehavioral assessments of memory function. Hypothesis: Focal ischemic stroke increases soluble amyloid beta (Aβ) in the hippocampus, causing impaired plasticity and memory function. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 30 days after recovery from transient MCAO (45 min) in adult (6-8 week) mice. A behavioral fear conditioning paradigm (CFC) was used to evaluate memory. ELISA assay was used to quantify soluble Aβ42 from the hippocampus. Slices were treated with Aβ42 oligomers with and without our newly developed peptide inhibitor of TRPM2, termed tatM2NX. Results: Recordings from brain slices 30 days after MCAO showed near complete loss of LTP; 161±9%, n=6 in sham compared to 115±4%, n=7 30 days after MCAO in the hippocampus. MCAO decreased freezing behavior, indicating lack of memory (65±7% in sham mice (n=6) and 37±7% in MCAO mice, n=7). We observed a 48% increase in Aβ42 in the hippocampus 30 days after MCAo. We observed that addition of Aβ42 oligomers (500 nM) impaired LTP. This impaired LTP was prevented with co-application of the TRPM2 channel inhibitor tatM2NX. Consistent with a role of TRPM2 channels in post-stroke cognitive impairment, MCAO mice treated with tatM2NX (20 mg/kg iv injection 24 hr before testing) on day 29 post MCA demonstrated increasing freezing to 72±5% (n=9). Conclusion: Our data implicates increased levels of soluble Aβ42 in the hippocampus following stroke, resulting in activation of TRPM2 channels and impaired synaptic plasticity. Therefore, reducing soluble Aβ42 and/or inhibition of TRPM2 channels at chronic time points following ischemia may represent a novel strategy to improve functional recovery following stroke.