Hope for vascular cognitive impairment: Ac-YVAD-cmk as a novel treatment against white matter rarefaction.

Vascular cognitive impairment (VCI) is the second leading cause of dementia with limited treatment options, characterised by cerebral hypoperfusion-induced white matter rarefaction (WMR). Subcortical VCI is the most common form of VCI, but the underlying reasons for region susceptibility remain elusive. Recent studies employing the bilateral cortical artery stenosis (BCAS) method demonstrate that various inflammasomes regulate white matter injury and blood-brain barrier dysfunction but whether caspase-1 inhibition will be beneficial remains unclear. To address this, we performed BCAS on C57/BL6 mice to study the effects of Ac-YVAD-cmk, a caspase-1 inhibitor, on the subcortical and cortical regions. Cerebral blood flow (CBF), WMR, neuroinflammation and the expression of tight junction-related proteins associated with blood-brain barrier integrity were assessed 15 days post BCAS. We observed that Ac-YVAD-cmk restored CBF, attenuated BCAS-induced WMR and restored subcortical myelin expression. Within the subcortical region, BCAS activated the NLRP3/caspase-1/interleukin-1beta axis only within the subcortical region, which was attenuated by Ac-YVAD-cmk. Although we observed that BCAS induced significant increases in VCAM-1 expression in both brain regions that were attenuated with Ac-YVAD-cmk, only ZO-1 and occludin were observed to be significantly altered in the subcortical region. Here we show that caspase-1 may contribute to subcortical regional susceptibility in a mouse model of VCI. In addition, our results support further investigations into the potential of Ac-YVAD-cmk as a novel treatment strategy against subcortical VCI and other conditions exhibiting cerebral hypoperfusion-induced WMR.

Anti-malarial drug, artemisinin and its derivatives for the treatment of respiratory diseases.

Artemisinins are sesquiterpene lactones with a peroxide moiety that are isolated from the herb Artemisia annua. It has been used for centuries for the treatment of fever and chills, and has been recently approved for the treatment of malaria due to its endoperoxidase properties. Progressively, research has found that artemisinins displayed multiple pharmacological actions against inflammation, viral infections, and cell and tumour proliferation, making it effective against diseases. Moreover, it has displayed a relatively safe toxicity profile. The use of artemisinins against different respiratory diseases has been investigated in lung cancer models and inflammatory-driven respiratory disorders. These studies revealed the ability of artemisinins in attenuating proliferation, inflammation, invasion, and metastasis, and in inducing apoptosis. Artemisinins can regulate the expression of pro-inflammatory cytokines, nuclear factor-kappa B (NF-κB), matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), promote cell cycle arrest, drive reactive oxygen species (ROS) production and induce Bak or Bax-dependent or independent apoptosis. In this review, we aim to provide a comprehensive update of the current knowledge of the effects of artemisinins in relation to respiratory diseases to identify gaps that need to be filled in the course of repurposing artemisinins for the treatment of respiratory diseases. In addition, we postulate whether artemisinins can also be repurposed for the treatment of COVID-19 given its anti-viral and anti-inflammatory properties.

A novel population of local pericyte precursor cells in tumor stroma that require Notch signaling for differentiation.

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1(+) precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146(+)/CD45(-)/CD31(-/lo) pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1(hi)/CD146(-)/CD45(-)/CD31(-). The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1(-/lo)/CD146(-)/CD45(-)/CD31(-) cells. Our results suggest that a local Sca1(hi)/CD146(-)/CD45(-)/CD31(-) pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes.

Endothelial-specific Notch blockade inhibits vascular function and tumor growth through an eNOS-dependent mechanism.

Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels.

Notch activation promotes endothelial survival through a PI3K-Slug axis.

RATIONALE: Loss of endothelial viability correlates with initiation and progress of vascular pathology. However, much remains to be learned about pathways required to maintain the balance between cell viability and apoptosis. Notch activation can enhance or inhibit apoptosis but its role in maintaining the endothelium needs further delineation. OBJECTIVE: This study aims to identify the mechanisms by which Notch activation regulates endothelial viability. METHODS AND RESULTS: Endothelial cells transduced with active Notch were treated with lipopolysaccharide (LPS) or homocysteine to induce endothelial apoptosis. Notch protected against LPS-induced cell death but exacerbated homocysteine-induced apoptosis. Inhibition of PI3K revealed that ligand-induced activation of endogenous Notch initiates parallel death and survival pathways and exhibits a differential effect on endothelial survival depending on the apoptotic stimulus. PI3K activity regulated the expression of Slug, which was required for survival in Notch-activated endothelial cells. Homocysteine, but not LPS, blocked both PI3K activity and Slug expression in Notch-activated cells, leading to increased endothelial apoptosis. CONCLUSIONS: Notch signaling leads to activation of parallel survival and apoptotic pathways in endothelial cells. The interaction of Notch with other signaling pathways plays an important contextual role in regulating endothelial viability.

Heterogeneity of breast cancer stem cells as evidenced with Notch-dependent and Notch-independent populations.

Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors, but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue, we blocked Notch activity in MCF-7 cells by expressing a dominant-negative MAML-GFP (dnMAML) construct, which inhibits signaling through all Notch receptors, and quantified the effect on tumor-initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor-initiating cell numbers in vivo showed a significant decrease, but not a complete abrogation, of these cells in dnMAML-expressing cells. Interestingly, when assessed in secondary assays in vitro or in vivo, there was no difference in tumor-initiating activity between the dnMAML-expressing cells and control cells. The fact that a subpopulation of dnMAML-expressing cells was capable of forming primary and secondary tumors indicates that there are Notch-independent tumor-initiating cells in the breast cancer cell line MCF-7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool, which will require combination therapies against multiple oncogenic pathways to eliminate the tumor-initiating cell population.

Heterotrimeric Gi/Go proteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway.

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-κB and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (Gα(i/o)) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. Gα(i/o) are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-κB. We find that Gα(i/o)-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.

Smooth Muscle alpha-actin is a direct target of Notch/CSL.

Intercellular signaling mediated by Notch receptors is essential for proper cardiovascular development and homeostasis. Notch regulates cell fate decisions that affect proliferation, survival, and differentiation of endothelial and smooth muscle cells. It has been reported that Jagged1-Notch interactions may participate in endocardial cushion formation by inducing endothelial-to-mesenchymal transformation. Here, we show that Notch directly regulates expression of the mesenchymal and smooth muscle cell marker smooth muscle alpha-actin (SMA) in endothelial and vascular smooth muscle cells via activation of its major effector, CSL. Notch/CSL activation induces SMA expression during endothelial-to-mesenchymal transformation, and Notch activation is required for expression of SMA in vascular smooth muscle cells. CSL directly binds a conserved cis element in the SMA promoter, and this consensus sequence is required for Notch-mediated SMA induction. This is the first evidence of the requirement for Notch activation in the regulation of SMA expression.

Identification of sokotrasterol sulfate as a novel proangiogenic steroid.

The potential to promote neovascularization in ischemic tissues using exogenous agents has become an exciting area of therapeutics. In an attempt to identify novel small molecules with angiogenesis promoting activity, we screened a library of natural products and identified a sulfated steroid, sokotrasterol sulfate, that induces angiogenesis in vitro and in vivo. We show that sokotrasterol sulfate promotes endothelial sprouting in vitro, new blood vessel formation on the chick chorioallantoic membrane, and accelerates angiogenesis and reperfusion in a mouse hindlimb ischemia model. We demonstrate that sulfation of the steroid is critical for promoting angiogenesis, as the desulfated steroid exhibited no endothelial sprouting activity. We thus developed a chemically synthesized sokotrasterol sulfate analog, 2beta,3alpha,6alpha-cholestanetrisulfate, that demonstrated equivalent activity in the hindlimb ischemia model and resulted in the generation of stable vessels that persisted following cessation of therapy. The function of sokotrasterol sulfate was dependent on cyclooxygenase-2 activity and vascular endothelial growth factor induction, as inhibition of either cyclooxygenase-2 or vascular endothelial growth factor blocked angiogenesis. Surface expression of alpha(v)beta(3) integrin was also necessary for function, as neutralization of alpha(v)beta(3) integrin, but not beta(1) integrin, binding abrogated endothelial sprouting and antiapoptotic activity in response to sokotrasterol sulfate. Our findings indicate that sokotrasterol sulfate and its analogs can promote angiogenesis in vitro and in vivo and could potentially be used for promoting neovascularization to relieve the sequelae of vasoocclusive diseases.

Proteomic identification of Ku70/Ku80 autoantigen recognized by monoclonal antibody against hepatocellular carcinoma.

A murine monoclonal antibody (mAb), CLD3 (IgG(1),kappa), was generated against hepatocellular carcinoma (HCC). Both immunofluorescence and immunohistochemical assays indicated the reactivity of CLD3 mAb localized at the nucleus and/or cytoplasm of tumorigenic HCC cell lines as well as in liver cancer tissues. By immunoprecipitation and using the matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach, the antigenic specificity of CLD3 was determined to be heterodimeric Ku70 and Ku80 autoantigen, which was confirmed by Western blotting.

Notch4-induced inhibition of endothelial sprouting requires the ankyrin repeats and involves signaling through RBP-Jkappa.

Notch proteins comprise a family of transmembrane receptors. Ligand activation of Notch releases the intracellular domain of the receptor that translocates to the nucleus and regulates transcription through the DNA-binding protein RBP-Jkappa. Previously, it has been shown that the Notch4 intracellular region (N4IC) can inhibit endothelial sprouting and angiogenesis. Here, N4IC deletion mutants were assessed for their ability to inhibit human microvascular endothelial cell (HMEC) sprouting with the use of a quantitative endothelial sprouting assay. Deletion of the ankyrin repeats, but not the RAM (RBP-Jkappa associated module) domain or C-terminal region (CT), abrogated the inhibition of fibroblast growth factor 2 (FGF-2)- and vascular endothelial growth factor (VEGF)-induced sprouting by Notch4, whereas the ankyrin repeats alone partially blocked sprouting. The ankyrin repeats were also the only domain required for up-regulation of RBP-Jkappa-dependent gene expression. Interestingly, enforced expression of the ankyrin domain alone was sufficient to up-regulate some, but not all, RBP-Jkappa-dependent genes. Although N4IC reduced VEGF receptor-2 (VEGFR-2) and vascular endothelial (VE)-cadherin expression, neither of these events is necessary and sufficient to explain N4IC-mediated inhibition of sprouting. A constitutively active RBP-Jkappa mutant significantly inhibited HMEC sprouting but not as strongly as N4IC. Thus, Notch4-induced inhibition of sprouting requires the ankyrin repeats and appears to involve RBP-Jkappa-dependent and -independent signaling.

Lipopolysaccharide initiates a TRAF6-mediated endothelial survival signal.

Similar to tumor necrosis factor (TNF), bacterial lipopolysaccharide (LPS) elicits parallel apoptotic and antiapoptotic pathways in endothelial cells. The overall result is that there is minimal endothelial cell death in response to LPS without inhibition of the cytoprotective pathway. While the TNF-induced death and survival pathways have been relatively well elucidated, much remains to be learned about LPS signaling events in this regard. It is known that the transcription factor nuclear factor-kappaB (NF-kappaB) provides a critical cell survival signal in response to TNF, but is not an essential component of the LPS-induced survival pathway. The TNF receptor-associated factor 6 (TRAF6) is a major effector of multiple LPS-induced signals, including a c-Jun N-terminal kinase (JNK)-mediated apoptotic response. In this report we demonstrate that following LPS stimulation, TRAF6 also transmits an important endothelial cell survival signal in a situation of complete NF-kappaB blockade. In response to LPS, TRAF6 activates the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway, but not ERK1/2 mitogen-activated protein kinases (MAPKs) in endothelial cells. Activation of PI3K signals a critical antiapoptotic pathway in response to LPS in endothelial cells, whereas ERK1/2 does not. Thus TRAF6 acts as a bifurcation point of the LPS-initiated death and survival signals in endothelial cells.

Notch4 inhibits endothelial apoptosis via RBP-Jkappa-dependent and -independent pathways.

Notch4, a member of the Notch family of transmembrane receptors, is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions, partly by regulating the propensity of cells to live or die. Various studies have demonstrated a role for Notch1 in modulating apoptosis, either in a positive or negative manner. In this study, we determined that constitutively active Notch4 (Notch4 intracellular domain) inhibited endothelial apoptosis triggered by lipopolysaccharide. Notch signals are transmitted by derepression and coactivation of the transcriptional repressor, RBP-Jkappa, as well as by less well defined mechanisms that are independent of RBP-Jkappa. A Notch mutant lacking the N-terminal RAM domain showed only partial antiapoptotic activity relative to Notch4 intracellular domain but stimulated equivalent RBP-Jkappa-dependent transcriptional activity. Similarly, constitutively active RBP-Jkappa activated a full transcriptional response but only demonstrated partial antiapoptotic activity. Additional studies suggest that Notch4 provides endothelial protection in two ways: inhibition of the JNK-dependent proapoptotic pathway in an RBP-Jkappa-dependent manner and induction of an antiapoptotic pathway through an RBP-Jkappa-independent up-regulation of Bcl-2. Our findings demonstrate that Notch4 activation inhibits apoptosis through multiple pathways and provides one mechanism to explain the remarkable capacity of endothelial cells to withstand apoptosis.

Bacterial lipopolysaccharide directly induces angiogenesis through TRAF6-mediated activation of NF-kappaB and c-Jun N-terminal kinase.

The intracellular pathways by which inflammatory mediators transmit their angiogenic signals is not well studied. The effects of a potent inflammatory mediator, bacterial lipopolysaccharide (LPS), are transmitted through Toll-like receptors (TLRs). A major, although not exclusive, LPS/TLR intracellular signaling pathway is routed through TNF (tumor necrosis factor) receptor associated factor 6 (TRAF6). In this report we demonstrate that LPS directly stimulates endothelial sprouting in vitro. By blocking TRAF6 activity using retroviral expression of a dominant-negative TRAF6 in endothelial cells, we show that TRAF6 is absolutely required for the LPS-initiated angiogenic response in vitro and in vivo. Inhibition of either c-Jun N-terminal kinase (JNK) activity or nuclear factor kappaB (NF-kappaB) activity, downstream of TRAF6, is sufficient to inhibit LPS-induced endothelial sprouting. In contrast, only inhibition of NF-kappaB, but not JNK, activity blocks basic fibroblast growth factor (bFGF)-induced angiogenesis. Our findings thus demonstrate a direct endothelial-stimulatory role of LPS in initiating angiogenesis through activation of TRAF6-dependent signaling pathways.

Trocarin, a blood coagulation factor Xa homologue from snake venom, causes inflammation and mitogenesis.

Trocarin, a Group D prothrombin activator from Tropidechis carinatus snake venom, has high sequence similarity to blood coagulation factor Xa (FXa). Both trocarin and FXa activate prothrombin to mature thrombin and have similar requirements for cofactors, such as factor Va, Ca2+ ions and phospholipids. In addition to its hemostatic functions, human FXa causes inflammation and induces mitogenesis in several cell types due to its interaction with effector protease receptor-1 (EPR-1). The inter-EGF domain region (L83FTKRL88) of FXa implicated in EPR-1-binding is distinctly different in trocarin (K83VLYQS88). Here we show that, interestingly, trocarin also causes edema in the mouse footpad; the inflammation, accompanied by a large purplish clot, is more persistent than the transient edema caused by FXa. Histological examination indicates significant differences between edema induced by FXa and trocarin. Moreover, trocarin-induced edema is not inhibited by a synthetic peptide based on the FXa-binding region of EPR-1, indicating that the inflammation is probably mediated by a mechanism independent of EPR-1-binding. Trocarin, like FXa, also has a mitogenic effect on bronchial smooth muscle cells mediated by an EPR-1-independent mechanism. Hence trocarin, being closely related to FXa, has similar non-hemostatic functions in mediating inflammation and mitogenesis, yet appears to act by distinctly different mechanisms.

Lipopolysaccharide signals an endothelial apoptosis pathway through TNF receptor-associated factor 6-mediated activation of c-Jun NH2-terminal kinase.

Inflammatory mediators such as TNF and bacterial LPS do not cause significant apoptosis of endothelial cells unless the expression of cytoprotective genes is blocked. In the case of TNF, the transcription factor NF-kappaB conveys an important survival signal. In contrast, even though LPS can also activate NF-kappaB, this signal is dispensable for LPS-inducible cytoprotective activity. LPS intracellular signals are transmitted through a member of the Toll-like receptor family, TLR4. This family of receptors transduces signals through a downstream molecule, TNFR-associated factor 6 (TRAF6). In this study, we demonstrate that the C-terminal fragment of TRAF6 (TRAF6-C) inhibits LPS-induced NF-kappaB nuclear translocation and c-Jun NH(2)-terminal kinase (JNK) activation in endothelial cells. In contrast, LPS activation of p38 kinase is not inhibited by TRAF6-C. TRAF6-C also inhibits LPS-initiated endothelial apoptosis, but potentiates TNF-induced apoptosis. LPS-induced loss of mitochondrial transmembrane potential, cytochrome c release, and caspase activation are all blocked by TRAF6-C. We demonstrate that TRAF6 signals apoptosis via JNK activation, since inhibition of JNK activation using a dominant-negative mutant also inhibits apoptosis. JNK inhibition blocks caspase activation, but the reverse is not true. Hence, JNK activation lies upstream of caspase activation in response to LPS stimulation.

Activated Notch4 inhibits angiogenesis: role of beta 1-integrin activation.

Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased beta1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of beta 1-integrins. Rather, we demonstrate that Notch4-expressing cells display beta1-integrin in an active, high-affinity conformation. Furthermore, using function-activating beta 1-integrin antibodies, we demonstrate that activation of beta1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting beta 1-integrin-mediated adhesion to the underlying matrix.