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The formation of intracranial aneurysm (IA) is associated with the destruction of various cellular and structural components, which induces pathogenic inflammatory responses that further propagate tissue damage. The regulatory immune system can suppress exacerbated inflammation and offer tissue protection; however, previous studies by others and us have demonstrated that the regulatory T (Treg) cells were functionally impaired in IA patients. Hence, strategies that can improve Treg function in IA patients should be investigated. Based on our previous finding that IL-2 strongly elevated the expression of the checkpoint molecule Tim-3 in Treg cells, we examined the effect of IL-2 in the function of Treg cells from IA patients. External IL-2 significantly improved the proliferation of Treg cells, increased the expression of CTLA-4 and LAG-3, and enhanced Treg-mediated suppression of conventional T cell (Tconv) proliferation. Importantly, compared to the Tim-3- Treg cells, the Tim-3+ Treg cells presented comparable proliferation capacity, but significantly greater expressions of CTLA-4 and LAG-3 and significantly higher capacity to suppress Tconv proliferation. In addition, blocking Tim-3 abrogated IL-2-mediated enhancement of Tim-3+ Treg cells. We then investigated the IL-2 level in IA patients, and found that although IA patients and healthy controls presented similar serum IL-2 concentration, the concentrations of IL-1β and TNF-α were significantly higher in IA patients than in healthy controls, signaling a relative reduction in IL-2 abundance. Together, we found that IL-2 could significantly enhance the function of Treg cells from IA patients in a Tim-3-dependent manner.

Pathogenic inflammation contributes to aneurysm formation by mediating the destruction of the endothelium and the extracellular matrix and promoting pathogenic proliferation of smooth muscle cells. In mouse models, tolerance-inducing T regulatory (Treg) cells could significantly reduce the incidence and severity of aneurysms. Hence, it should be investigated why in human intracranial aneurysm (IA) patients, Treg cells failed to provide protection against aneurysm formation. In this study, the frequency and function of Treg cells in IA patients were examined. The frequency of Foxp3+ Treg cells was significantly lower in IA patients than in healthy controls. This downregulation was only specific to the Treg subset of CD4+ T cells, as the frequency of total CD4+ T cell was increased in IA patients. Subsequently, we found that the expressions of Treg-associated molecules, including Foxp3, CTLA-4, TGF-β, and IL-10, were significantly lower in Foxp3+ Treg cells from IA patients than in Foxp3+ Treg cells from healthy controls. In both healthy controls and IA patients, Foxp3+ Treg cells were distinguished into a more potent Tim-3+ subset and a less potent Tim-3− subset. The Tim-3+ subset of Foxp3+ Treg cells was significantly reduced in IA patients. Signaling via IL-2, IL-7, IL-15 and IL-21 was shown to promote Tim-3 upregulation in CD4+ and CD8+ T cells. Interestingly, we found that Tim-3 could be upregulated in Treg cells via the same mechanism, but compared to the Treg cells from healthy controls, the Treg cells from IA patients presented defects in Tim-3 upregulation upon cytokine stimulation. Together, our results demonstrated that Foxp3+ Treg cells in IA patients presented reduced function, which was associated with a defect in Tim-3 upregulation.

In this report, we describe an efficient and non-enzymatic method for isolating and culturing endothelial cells (ECs) from the nidus of surgically resected arteriovenous malformation (AVM) specimens. These cultured cells possessed typical phenotypic markers (i.e. von Willebrand factor and CD34), as well as morphological and ultrastructural characteristics of ECs. However, they had activated Notch-1 signaling, which plays a critical role in the development of AVM. The present study suggests that hypoxic endothelial cells from the nidus of human cerebral arteriovenous malformation (CAVMECs) have angiogenic potentials, as our data showed that VEGF gene expression and cell proliferation were more evident with prolonged hypoxia. In our study, we successfully used the vascular tissue explants adherent method to isolate and culture CAVMECs with high purity. This may prove to be a useful tool for studying the molecular mechanisms that mediate abnormal vessel development and maintenance in AVM.

The study was performed to investigate the effect of combination therapy with melatonin and dexamethasone (DEX) on brain injury in intracerebral hemorrhage (ICH) model in rats. Compared to the control group, combination therapy group with melatonin (10 mg/kg) and DEX (0.025 mg/kg) significantly reduced the degree of (1) brain edema, (2) the permeability of blood brain barrier (measured by Evans blue), (3) Oxidative stress (evaluated by malondialdehyde assay), (4) cytokines expression (tumor necrosis factor-alpha and interleukin-1beta), and (5) apoptosis (measured by Bax and Bcl-2 expression). In addition, we have also clearly demonstrated that the combination therapy significantly ameliorated neurologic scores. Taken together, our results clearly indicated for the first time that strategies targeting multiple proinflammatory pathways may be more effective than a single effector molecule for the treatment of ICH.