Ciliogenesis Mechanisms Mediated by PAK2-ARL13B Signaling in Brain Endothelial Cells is Responsible for Vascular Stability

In the developing vasculature, cilia, microtubule-based organelles that project from the apical surface of endothelial cells (ECs), have been identified to function cell autonomously to promote vascular integrity and prevent hemorrhage. To date, the underlying mechanisms of endothelial cilia formation (ciliogenesis) are not fully understood. Understanding these mechanisms is likely to open new avenues for targeting EC-cilia to promote vascular stability. Here, we hypothesized that brain ECs ciliogenesis and the underlying mechanisms that control this process are critical for brain vascular stability. To investigate this hypothesis, we utilized multiple approaches including developmental zebrafish model system and primary cell culture systems. In the p21 activated kinase 2 (pak2a) zebrafish vascular stability mutant [redhead (rhd)] that shows cerebral hemorrhage, we observed significant decrease in cilia-inducing protein ADP Ribosylation Factor Like GTPase 13B (Arl13b), and a 4-fold decrease in cilia numbers. Overexpressing ARL13B-GFP fusion mRNA rescues the cilia numbers (1–2-fold) in brain vessels, and the cerebral hemorrhage phenotype. Further, this phenotypic rescue occurs at a critical time in development (24 h post fertilization), prior to initiation of blood flow to the brain vessels. Extensive biochemical mechanistic studies in primary human brain microvascular ECs implicate ligands platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor. Thus, collectively, we have implicated a critical brain ECs ciliogenesis signal that converges on PAK2-ARL13B proteins to promote vascular stability

Cilia Proteins are Biomarkers of Altered Flow in the Vasculature

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation—the physical removal of cilia from the cell surface—is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium

Cilia proteins are biomarkers of altered flow in the vasculature

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation- the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.This work was funded by Qatar National Research Fund, National Priority Research Program (NPRP 10-0123-170222 to HCY). ADS is supported by funds from the Department of Pediatrics, Herma Heart Institute, the National Center for Research Resources, and the National Center for Advancing Translational Sciences, NIH (UL1TR001436)

RDEA119 enhances the percentage of SSRBCs circulating in bloodstream.

<p>Anesthetized mice were injected with fluorescence-labeled sham- (▪) or fluorescence-labeled RDEA119-treated SSRBCs (□) (n = 3 for each treatment). Blood samples were collected from sham-treated and RDEA119-treated SSRBCs after 1, 10 and 20 min of human SSRBC infusion. Error bars show SEM of three different experiments. A significantly greater percentage of RDEA119-treated than sham-treated SSRBCs was retained in the circulation at 10 and 20 min post SSRBC infusion. *: <i>p</i><0.05 compared to sham-treated SSRBCs.</p

MEK inhibition down-regulates SSRBC adhesion to both non-activated and activated endothelial cells <i>in vitro</i>.

<p>The effects of MEK inhibitors on SSRBC adhesion to HUVECs, HMVECs-d and EOMA cells was tested in intermittent flow condition assays at different shear stresses <i>in vitro</i>. Results are presented as % adherent SSRBCs at a shear stress of 2 dynes/cm². <b>A.</b> SSRBCs were sham-treated or treated with 100 nM MEK inhibitor U0126 prior to adhesion assays to non-treated and TNFα-treated HUVECs. *: <i>p</i><0.0001 compared to sham-treated SSRBCs adherent to non-treated HUVECs; **: <i>p</i><0.001 compared to sham-treated SSRBCs adherent to non-treated HUVECs; ***: <i>p</i><0.0001 compared to sham-treated SSRBCs adherent to TNFα-treated HUVECs. Error bars show standard error mean (SEM) of 4 different experiments. <b>B.</b> SSRBCs were sham-treated, or treated with 100 nM RDEA119, 100 nM AZD6244, 100 nM trametinib, or 10 µM damnacanthal prior to adhesion assays to non-treated and TNFα-treated HUVECs. *: <i>p</i><0.0001 compared to sham-treated SSRBCs adherent to non-treated HUVECs; **: <i>p</i><0.0001 compared to sham-treated SSRBCs adherent to TNFα-treated HUVECs; and ^†: <i>p</i><0.001 compared to sham-treated SSRBCs adherent to non-treated HUVECs. Error bars show SEM of 3 different experiments. <b>C</b>–<b>D.</b> SSRBCs and normal RBCs (AARBCs) were sham-treated, or treated with 100 nM U0126 or 100 nM RDEA119 prior to adhesion assays to non-treated and TNFα-treated HMVECs-d (<b>C</b>) and EOMA cells (<b>D</b>). *: <i>p</i><0.0001 compared to sham-treated AARBCs adherent to non-treated HMVECs-d (<b>C</b>) and EOMA cells (<b>D</b>); **: <i>p</i><0.0001 compared to sham-treated SSRBCs adherent to non-treated HMVECs-d (<b>C</b>) and EOMA cells (<b>D</b>); and ***: <i>p</i><0.001 compared to sham-treated SSRBCs adherent to TNFα-treated HMVECs-d (<b>C</b>) and EOMA cells (<b>D</b>). Error bars show SEM of 3 different experiments for <b>C</b> and <b>D</b>.</p

The MEK inhibitor U0126 abrogates SSRBC adhesion and vasoocclusion <i>in vivo</i>.

<p>Nude mice implanted with dorsal skin-fold window chambers were injected with murine TNFα. Four hours later, intravital microscopic observations of post-capillary venules using 10× and 20× magnifications were conducted through the window chamber immediately after infusion of fluorescently-labeled sham- (<b>A</b>; panels 1, 2 and 3) or U0126-treated (<b>B</b>; panels 1, 2 and 3) human SSRBCs (n = 5). SSRBC adhesion and vasoocclusion are indicated with arrows. While sham-treated SSRBCs adhered markedly to venule walls promoting vasoocclusion, U0126 treatment of SSRBCs significantly reduced SSRBC adhesion and stasis. Scale bar = 50 µm. <b>C.</b> Video frames showing vessel segments were used to quantify adhesion in venules of animals occupied by SSRBCs (n = 5 for each treatment). Adhesion of fluorescently labeled sham-treated SSRBCs (sham-treated SS) and U0126-treated SSRBCs (U0126-treated SS) observed in all vessels recorded presented as fluorescence intensity of adherent SSRBCs (pixels). <b>D</b>–<b>F.</b> The values of at least 35 segments of vessels were analyzed and averaged among groups of animals (n = 5) to represent percentage of vessels occupied by adherent SSRBCs (<b>D</b>); percentage of vessels with normal blood flow, slow blood flow and no blood flow (<b>E</b>); and percentage of normal flowing vessels (<b>F</b>). Error bars show SEM of 5 different experiments for each treatment condition. *: <i>p</i> = 0.0103 (<b>C</b>) and <i>p</i><0.0001 (<b>D</b> and <b>F</b>) compared to sham-treated SS regardless of the vessel diameter within the ranges specified.</p

Oxidative Stress and Thrombosis during Aging: The Roles of Oxidative Stress in RBCs in Venous Thrombosis

Mid-life stage adults are at higher risk of developing venous thrombosis (VT)/thromboembolism (VT/E). Aging is characterized by an overproduction of reactive oxygen species (ROS), which could evoke a series of physiological changes involved in thrombosis. Here, we focus on the critical role of ROS within the red blood cell (RBC) in initiating venous thrombosis during aging. Growing evidence has shifted our interest in the role of unjustifiably unvalued RBCs in blood coagulation. RBCs can be a major source of oxidative stress during aging, since RBC redox homeostasis is generally compromised due to the discrepancy between prooxidants and antioxidants. As a result, ROS accumulate within the RBC due to the constant endogenous hemoglobin (Hb) autoxidation and NADPH oxidase activation, and the uptake of extracellular ROS released by other cells in the circulation. The elevated RBC ROS level affects the RBC membrane structure and function, causing loss of membrane integrity, and decreased deformability. These changes impair RBC function in hemostasis and thrombosis, favoring a hypercoagulable state through enhanced RBC aggregation, RBC binding to endothelial cells affecting nitric oxide availability, RBC-induced platelet activation consequently modulating their activity, RBC interaction with and activation of coagulation factors, increased RBC phosphatidylserine exposure and release of microvesicles, accelerated aging and hemolysis. Thus, RBC oxidative stress during aging typifies an ultimate mechanism in system failure, which can affect major processes involved in the development of venous thrombosis in a variety of ways. The reevaluated concept of the critical role of RBC ROS in the activation of thrombotic events during aging will help identify potential targets for novel strategies to prevent/reduce the risk for VT/E or VT/E recurrences in mid-life stage adults

The MEK inhibitor RDEA119 diminishes human SSRBC organ infiltration and trapping.

<p>Nude mice preinjected with murine TNFα were infused 4 hours later with sham-treated SSRBCs (sham-treated SS) or RDEA119-treated SSRBCs (RDEA119-treated SS) (50% hematocrit; n = 3 for each treatment). <b>A and B</b>. Two hours following RBC infusion, animals were sacrificed and the lungs, liver, spleen and kidneys were harvested. Tissue sections were analyzed and quantitated for the presence of fluorescently labeled SSRBCs. The three panels for each treatment in <b>A</b> represent three different experiments with similar results. Scale bar = 150 µm. <b>B.</b> The effect of RDEA119 treatment on SSRBC trapping in organs was quantitated and presented as fluorescence intensity of fluorescence-labeled SSRBC trapped in organs (pixels). RDEA119 treatment had a significant effect on trapping of SSRBCs in the lungs, liver and spleen compared to sham-treated cells. *: <i>p</i><0.001 compared to sham-treated SSRBCs. Error bars show SEM of three different experiments.</p

MEK inhibition prevents SSRBCs from activating neutrophil adhesion.

<p>The effect of MEK inhibitors on the ability of SSRBCs to stimulate neutrophil (PMN) adhesion to ECs was tested. <b>A</b> and <b>B</b>. SSRBCs (n = 8) were sham-treated or treated with 100 nM MEK inhibitor U0126, RDEA119, AZD6244 or trametinib. Washed treated SSRBCs were then co-incubated with ABO-matched naïve PMNs isolated from healthy donors (n = 8), prior to testing adhesion of PMNs to HUVECs (<b>A;</b> n = 4) and HMVECs-d (<b>B;</b> n = 4) in intermittent flow condition assays at different shear stresses. <b>C.</b> AARBCs (n = 3) were sham-treated, washed, and then co-incubated with ABO-matched naïve PMNs isolated from healthy donors (n = 3), prior to testing adhesion of PMNs to HUVECs and HMVECs-d at different shear stresses. <b>D.</b> Non-treated and TNFα-treated HUVECs were co-incubated with sham-treated SSRBCs, U0126-treated SSRBCs or sham-treated AARBCs. HUVECs were then washed free of non-adherent RBCs, and tested for their ability to support adhesion of PMNs (n = 3). Results are presented as % adherent PMNs at a shear stress of 1 dyne/cm². *:<i>p</i><0.0001 compared to adhesion of naïve PMNs (PMNs only) to non-treated ECs; and **:<i>p</i><0.0001 compared to adhesion of PMNs stimulated with SSRBCs (PMNs+SSRBCs). Error bars show SEM of 4 different experiments for <b>A and B</b>, and 3 different experiments for <b>C</b> and <b>D</b>.</p