BBB Protection in HIV Infection: Barrier-shielding effects of PARP inhibition

描述（由申请人提供）：尽管引入了联合抗逆转录病毒治疗，但HIV相关神经认知障碍（HAND）的患病率仍然很高。HAND与血液中促炎因子和活化的受感染单核细胞亚群的升高相关，两者均显示引起有助于HAND的血脑屏障（BBB）损伤。破坏单核细胞迁移的治疗策略可以减缓HIV感染和BBB损伤的进展，从而改善HAND。在上一个资助期间，我们重点研究了（1）BBB损伤的分子机制和（2）糖原合成酶激酶（GSK）3 β抑制剂在HIV感染驱动的神经炎症中的抗炎和屏障保护特性。我们揭示了BBB功能障碍的分子机制[紧密连接（TJ）磷酸化，BBB处的CD 40/CD 40配体相互作用以及HIV对脑内皮直接影响背后的信号传导事件]。我们证明了由于TJ稳定化，在脑内皮中GSK 3 β抑制后屏障收紧。我们在体外和体内发现了GSK 3 β抑制在脑内皮中的有效抗炎作用（抑制单核细胞迁移、减少炎症因子产生和BBB保护）。单核细胞中的GSKl抑制减弱了它们穿过BBB的粘附/迁移，通过抑制小GTl、Rac 1下调活性整合素表达，并保护BBB。然而，BBB屏蔽特性或单核细胞迁移/粘附的抑制在体外或体内都没有完全达到，这表明补充GSK 3 β的其他途径对于BBB功能的恢复是必要的。为了寻找这样的分子，我们将注意力转向聚（ADP-核糖）聚合酶-1（PARP-1）及其抑制剂，最近被认为是具有显着神经保护特性的抗炎/免疫调节剂。基于初步数据，我们提出PARP抑制将通过对单核细胞、脑内皮细胞、活化的小胶质细胞和HIV-1感染的巨噬细胞的影响来减轻HIV-1引起的BBB损伤。事实上，初步数据表明，在原代人脑微血管内皮细胞（BMVEC）中的PARP抑制改善了BBB的完整性和增强了TJ蛋白的表达。PARP抑制剂可防止炎症因子引起的屏障破坏，减少单核细胞在BBB模型中的粘附/迁移，下调粘附分子/促炎分子，降低RhoA/Rac 1的活性。在单核细胞中，PARP抑制剂下调了抑制RhoA/Rac 1抑制的活性β-整联蛋白。 PARP抑制剂降低了促炎分子的表达，并减少了人类巨噬细胞中的HIV复制。虽然PARP抑制剂对免疫细胞的调节作用已在一定程度上进行了研究，但其在HIV CNS感染背景下的作用尚不清楚。PARP抑制剂现已进入癌症治疗的临床试验阶段，确保快速转化为免疫/炎症疾病的治疗。

DESCRIPTION (provided by applicant): Prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite the introduction of combined antiretroviral therapy. HAND is associated with elevation of pro-inflammatory factors in blood and subsets of activated infected monocytes, both shown to cause blood brain barrier (BBB) impairment that contributes to HAND. Therapeutic strategies that disrupt monocyte migration can slow progression of HIV infection and BBB injury, thereby ameliorating HAND. During the previous period of funding, we focused on studies of (1) molecular mechanisms of BBB injury and (2) anti-inflammatory and barrier protective properties of glycogen synthase kinase (GSK) 3ß inhibition in neuroinflammation driven by HIV infection. We uncovered molecular mechanisms of BBB dysfunction [tight junction (TJ) phosphorylation, CD40/CD40 ligand interactions at BBB and signaling events behind the direct effects of HIV on brain endothelium]. We demonstrated barrier tightening following GSK3ß suppression in brain endothelium due to TJ stabilization. We uncovered potent anti-inflammatory effects of GSK3ß inhibition in brain endothelium (suppression of monocyte migration, diminution of inflammatory factor production, and BBB protection) in vitro and in vivo. GSKß inhibition in monocytes attenuated their adhesion/migration across the BBB, down regulated active integrin expression via suppression of the small GTPase, Rac1, and protected the BBB. Yet, BBB shielding properties or inhibition of monocyte migration/adhesion were not fully attained in vitro or in vivo, suggesting that additional pathways complimentary to GSK3ß are necessary for the restitution of BBB function. In search of such molecules, we turned our attention to poly(ADP-ribose) polymerase-1 (PARP-1) and its inhibitors, recently recognized as anti-inflammatory/immune modulating agents with significant neuroprotective properties. Based upon preliminary data, we propose that PARP inhibition will attenuate BBB injury caused by HIV-1 via effects on monocytes, brain endothelium, activated microglia and HIV-1 infected macrophages. Indeed, preliminary data indicate that PARP suppression in primary human brain microvascular endothelial cells (BMVEC) improved BBB integrity and augmented expression of TJ proteins. PARP inhibitors prevented barrier disruption caused by inflammatory factors, diminished monocyte adhesion/migration across a BBB model, down regulated adhesion molecules/pro-inflammatory molecules and decreased activity of RhoA/Rac1. In monocytes, PARP inhibitors down regulated the active ß-integrin that paralleled RhoA/Rac1 suppression. PARP inhibitors decreased expression of pro-inflammatory molecules and diminished HIV replication in human macrophages. Although the modulatory effects of PARP inhibitors on immune cells have been studied to some extent, nothing is known about their effects in the setting of HIV CNS infection. PARP inhibitors have now reached the stage of clinical testing for cancer treatment, assuring quick translation to therapy of immune/inflammatory disorders.