CD31 as a probable responding and gate-keeping protein of the blood-brain barrier and the risk of Alzheimer's disease.

Several studies have shown that an abnormal vascular-immunity link could increase Alzheimer's disease (AD) risk; however, the mechanism is unclear. CD31, also named platelet endothelial cell adhesion molecule (PECAM), is a surface membrane protein of both endothelial and immune cells and plays important roles in the interaction between the vascular and immune systems. In this review, we focus on research regarding CD31 biological actions in the pathological process that may contribute to AD based on the following rationales. First, endothelial, leukocyte and soluble forms of CD31 play multi-roles in regulating transendothelial migration, increasing blood-brain barrier (BBB) permeability and resulting in neuroinflammation. Second, CD31 expressed by endothelial and immune cells dynamically modulates numbers of signaling pathways, including Src family kinases, selected G proteins, and ß-catenin which in turn affect cell-matrix and cell-cell attachment, activation, permeability, survival, and ultimately neuronal cell injury. In endothelia and immune cells, these diverse CD31-mediated pathways act as a critical regulator in the immunity-endothelia-brain axis, thereby mediating AD pathogenesis in ApoE4 carriers, which is the major genetic risk factor for AD. This evidence suggests a novel mechanism and potential drug target for CD31 in the background of genetic vulnerabilities and peripheral inflammation for AD development and progression.

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid's growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

Somatic PRKAR1A mutation in sporadic atrial myxoma with cerebral parenchymal metastases: a case report.

BACKGROUND: Atrial myxomas are generally considered benign neoplasms. The majority of tumors are sporadic and less than 10% are associated with an autosomal dominant condition known as the Carney complex, which is most often caused by germline mutation in the gene PRKAR1A. Whether this gene plays a role in the development of sporadic myxomas has been an area of debate, although recent studies have suggested that some fraction of sporadic tumors also carry mutations in PRKARIA. Extra-cardiac complications of atrial myxoma include dissemination of tumor to the brain; however, the dissemination of viable invasive tumor cells is exceedingly rare. CASE PRESENTATION: We present here a 48-year-old white woman who developed multiple intracranial hemorrhagic lesions secondary to tumor embolism that progressed to 'false' aneurysm formation and invasion through the vascular wall into brain parenchyma 7 months after resection of an atrial myxoma. Whole exome sequencing of her tumor revealed multiple mutations in PRKAR1A not found in her germline deoxyribonucleic acid (DNA), suggesting that the myxoma in this patient was sporadic. CONCLUSIONS: Our patient illustrates that mutations in PRKAR1A may be found in sporadic lesions. Whether the presence of this mutation affects the clinical behavior of sporadic tumors and increases risk for metastasis is not clear. Regardless, the protein kinase A pathway which is regulated by PRKAR1A represents a possible target for treatment in patients with metastatic cardiac myxomas harboring mutations in the PRKARIA gene.

Minocycline mitigates the effect of neonatal hypoxic insult on human brain organoids.

Neonatal hypoxic injury (NHI) is a devastating cause of disease that affects >60% of babies born with a very low birth weight, resulting in significant morbidity and mortality, including life-long neurological consequences such as seizures, cerebral palsy, and intellectual disability. Hypoxic injury results in increased neuronal death, which disrupts normal brain development. Although animal model systems have been useful to study the effects of NHI, they do not fully represent the uniqueness and complexities of the human brain. To better understand the effects of hypoxia on human brain development, we have generated a brain organoid protocol and evaluated these cells over the course of 6 months. As anticipated, the expression of a forebrain marker, FOXG1, increased and then remained expressed over time, while there was a transition in the expression of the deep-layer (TBR1) and upper-layer (SATB2) cortical markers. In addition, ventral genes (Eng1 and Nkx2.1) as well as markers of specialized nonneuronal cells (Olig2 and GFAP) also increased at later time points. We next tested the development of our in vitro cerebral organoid model at different oxygen concentrations and found that hypoxia repressed gene markers for forebrain, oligodendrocytes, glial cells, and cortical layers, as well as genes important for the migration of cortical neurons. In contrast, ventral markers were either unaffected or even increased in expression with hypoxic insult. Interestingly, the negative effect of hypoxia on the dorsal brain genes as well as oligodendrocytes, and neuronal progenitors could be mitigated by the use of minocycline, an FDA-approved small molecule. Taken together, we have generated a unique and relevant in vitro human brain model system to study diseases such as NHI as well as their potential treatments. Using this system, we have shown the efficacy of minocycline for human NHI.

MMP-2: A modulator of neuronal precursor activity and cognitive and motor behaviors.

Matrix Metalloproteinase2, (MMP2, gelatinase A) is a zinc-containing enzyme with a broad substrate specificity including components of the extracellular matrix, cell surface molecules and a wide range bioactive molecules. MMP2 is known to play important roles in a variety of signaling pathways and processes in a wide range of cell types and tissues. In this report we elucidate the effects of the absence of MMP2 in Neural Precursor Cells (NPC) derived from C57BL/6 MMP2 KO mice and in primary and secondary neurosphere formation. We observed smaller neurosphere numbers and sizes, decreased NPC numbers, PCNA expression, DNA and Akt activation in MMP2 KO NPC compared to WT NPC. We also found decreased neurosphere formation and NPC migration outward from adherent neurospheres, decreased CXCR4 and nestin expression and increased GFAP and neuro-filament expression in MMP2 KO NPC compared to Wt NPC. MMP2 KO mice were found to exhibit increased anxiety manifested in open field activity assays compared to Wt mice. MMP2 KO mice also exhibited differences in motor activities manifested by decreased balance and endurance during Rota-rod testing. These studies illustrate an important role of MMP2 in cognitive and motor behaviors and confirm its importance in NPC activities crucial to brain development, growth and response to and recovery from injury.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

OBJECTIVE: Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. APPROACH AND RESULTS: AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 µm; P=0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P=0.0015; interleukin-10: 7.6-fold, P=0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 µm; P=0.0306), increased collagen density (2.4-fold; P=0.0432), and increased number of M2 macrophages (2.1-fold; P=0.0335). CONCLUSIONS: CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation.

As human lung microvascular endothelia achieve confluence, src family kinases are activated, and tyrosine-phosphorylated p120 catenin physically couples NEU1 sialidase to CD31.

In postconfluent human pulmonary microvascular endothelial cell (HPMEC)s, NEU1 sialidase associates with and desialylates the src family kinase (SFK) substrate, CD31, and disrupts angiogenesis. We asked whether the NEU1-CD31 interaction might be SFK-driven. We found that normalized phospho-SFK (PY416) signal is increased in postconfluent HPMECs compared to subconfluent cells and prior SFK inhibition with PP2 or SU6656 completely blocked NEU1 association with and desialylation of CD31. Prior silencing of each of the four SFKs expressed in HPMECs, as well as CD31, dramatically reduced confluence-induced SFK activation. No increases in tyrosine phosphorylation of NEU1 or CD31 were detected. However, in postconfluent cells, we found increased tyrosine phosphorylation of a 120 kDa protein that was identified as p120 catenin (p120ctn). Prior silencing of c-src, fyn, or yes each reduced p120ctn phosphorylation. Prior knockdown of p120ctn prevented NEU1-CD31 association in both co-immunoprecipitation and pull-down assays. In these same assays, p120ctn associated with each of the four HPMEC-expressed SFKs as well as CD31 and NEU1. The CD31-p120ctn interaction was SFK-dependent whereas the NEU1-p120ctn interaction was not. Using purified recombinant binding partners in a cell-free system, direct protein-protein interactions between NEU1, CD31, and p120ctn were detected. Our combined data indicate that as HPMECs achieve confluence and CD31 ectodomains become homophilically engaged, multiple SFKs are activated to increase tyrosine phosphorylation of p120ctn, which in turn, functions as a cross-bridging adaptor molecule that physically couples NEU1 to CD31, permitting NEU1-mediated desialylation of CD31. These findings establish a SFK-driven, p120ctn-dependent mechanism for NEU1 recruitment to CD31.

The role of endothelial HIF-1 αin the response to sublethal hypoxia in C57BL/6 mouse pups.

Chronic sublethal hypoxia, a complication of premature birth, is associated with cognitive and motor handicaps. Responsiveness to and recovery from this hypoxic environment is dependent on induction of HIF-1 α in the cells affected. Microvascular endothelial-glial and microvascular endothelial-neuronal precursor interactions have been found to be dynamic and reciprocal, involving autocrine and paracrine signaling, with response and recovery correlated with baseline levels and levels of induction of HIF-1 α.To ascertain the roles of endothelial HIF-1 α in the responses of brain microvascular endothelial cells (EC) and neuronal precursors to hypoxia, we examined the effects of the presence and absence of endothelial HIF-1 α expression in culture and in cells comprising the subventricular zone (SVZ) and dentate gyrus under normoxic and hypoxic conditions. We used C57BL/6 WT and EC HIF-1 α -deficient mice and brain microvascular ECs isolated from these mice in western blots, immunofluorescence, and behavioral studies to examine the roles of EC HIF-1 α behaviors of endothelial and neuronal precursor cells (NPCs) in SVZ and hippocampal tissues under normoxic and hypoxic conditions and behaviors of these mice in open field activity tests. Analyses of ECs and SVZ and dentate gyrus tissues revealed effects of the absence of endothelial HIF-1 α on proliferation and apoptosis as well as open field activity, with both ECs and neuronal cells exhibiting decreased proliferation, increased apoptosis, and pups exhibiting gender-specific differences in open field activities. Our studies demonstrate the autocrine and paracrine effects of EC HIF-1 α-modulating proliferative and apoptotic behaviors of EC and NPC in neurogenic regions of the brain and gender-specific behaviors in normoxic and hypoxic settings.

-NOD Mice Having a Lyn Tyrosine Kinase Mutation Exhibit Abnormal Neutrophil Chemotaxis.

Neutrophils from NOD (Non-Obese Diabetic) mice exhibited reduced migration speed, decreased frequency of directional changes, and loss of directionality during chemotaxis (compared to wild-type [WT] C57BL/6 mice). Additionally, F-actin of chemotaxing NOD neutrophils failed to orient toward the chemoattractant gradient and NOD neutrophil adhesion was impaired. A point mutation near the autophosphorylation site of Lyn in NOD mice was identified. Point mutations of G to A (G1412 in LynA and G1199 in LynB) cause a change of amino acid E393 (glutamic acid) to K (lysine) in LynA (E393 →K) (E372 of LynB), affecting fMLP-induced tyrosine phosphorylation. These data indicate that the Lyn mutation in NOD neutrophils is likely responsible for dysregulation of neutrophil adhesion and directed migration, implying the role of Lyn in modulating diabetic patient's susceptibility to bacterial and fungal infections. J. Cell. Physiol. 232: 1689-1695, 2017. © 2016 Wiley Periodicals, Inc.

Targeted proteomics effectively quantifies differences between native lung and detergent-decellularized lung extracellular matrices.

Extracellular matrix is a key component of many products in regenerative medicine. Multiple regenerative medicine products currently in the clinic are comprised of human or xenogeneic extracellular matrix. In addition, whole-organ regeneration exploits decellularized native organs as scaffolds for organotypic cell culture. However, precise understanding of the constituents of such extracellular matrix-based implants and scaffolds has sorely lagged behind their use. We present here an advanced protein extraction method using known quantities of proteotypic 13C-labeled peptides to quantify matrix proteins in native and decellularized lung tissues. Using quantitative proteomics that produce picomole-level measurements of a large number of matrix proteins, we show that a mild decellularization technique ("Triton/SDC") results in near-native retention of laminins, proteoglycans, and other basement membrane and ECM-associated proteins. Retention of these biologically important glycoproteins and proteoglycans is quantified to be up to 27-fold higher in gently-decellularized lung scaffolds compared to scaffolds generated using a previously published decellularization regimen. Cells seeded onto this new decellularized matrix also proliferate robustly, showing positive staining for proliferating cell nuclear antigen (PCNA). The high fidelity of the gently decellularized scaffold as compared to the original lung extracellular matrix represents an important step forward in the ultimate recapitulation of whole organs using tissue-engineering techniques. This method of ECM and scaffold protein analysis allows for better understanding, and ultimately quality control, of matrices that are used for tissue engineering and human implantation. These results should advance regenerative medicine in general, and whole organ regeneration in particular. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) in large part defines the biochemical and mechanical properties of tissues and organs; these inherent cues make acellular ECM scaffolds potent substrates for tissue regeneration. As such, they are increasingly prevalent in the clinic and the laboratory. However, the exact composition of these scaffolds has been difficult to ascertain. This paper uses targeted proteomics to definitively quantify 71 proteins present in acellular lung ECM scaffolds. We use this technique to compare two decellularization methods and demonstrate superior retention of ECM proteins important for cell adhesion, migration, proliferation, and differentiation in scaffolds treated with low-concentration detergent solutions. In the long term, the ability to acquire quantitative biochemical data about biological substrates will facilitate the rational design of engineered tissues and organs based on precise cell-matrix interactions.

CD44 Influences Fibroblast Behaviors Via Modulation of Cell-Cell and Cell-Matrix Interactions, Affecting Survivin and Hippo Pathways.

CD44 has been studied in a wide variety of cell types, in a diverse array of cell behaviors and in a diverse range of signaling pathways. We now document a role for CD44 in mediating fibroblast behaviors via regulation of N-cadherin, extracellular matrix expression, Survivin and the Hippo pathway. Here, we report our findings on the roles of CD44 in modulating proliferation, apoptosis, migration and invasion of murine wild-type (WT-FB) and CD44 knockout dermal fibroblasts (CD44KO-FB). As we have documented in microvascular endothelial cells lacking CD44, we found persistent increased proliferation, reduced activation of cleaved caspase 3, increased initial attachment, but decreased strength of cell attachment in high cell density, post confluent CD44KO-FB cultures. Additionally, we found that siRNA knock-down of CD44 mimicked the behaviors of CD44KO-FB, restoring the decreases in N-cadherin, collagen type I, fibronectin, Survivin, nuclear fractions of YAP and phospho-YAP and decreased levels of cleaved caspase 3 to the levels observed in CD44KO-FB. Interestingly, plating CD44KO-FB on collagen type I or fibronectin resulted in significant decreases in secondary proliferation rates compared to plating cells on non-coated dishes, consistent with increased cell adhesion compared to their effects on WT-FB. Lastly, siRNA knockdown of CD44 in WT-FB resulted in increased fibroblast migration compared to WT-FB, albeit at reduced rates compared to CD44KO-FB. These results are consistent with CD44's pivotal role in modulating several diverse behaviors important for adhesion, proliferation, apoptosis, migration and invasion during development, growth, repair, maintenance and regression of a wide variety of mesenchymal tissues.

ENPP1-Fc prevents mortality and vascular calcifications in rodent model of generalized arterial calcification of infancy.

Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg's sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and common diseases of ectopic vascular calcification.

Modulation of Sox10, HIF-1α, Survivin, and YAP by Minocycline in the Treatment of Neurodevelopmental Handicaps following Hypoxic Insult.

Premature infants are at an increased risk of developing cognitive and motor handicaps due to chronic hypoxia. Although the current therapies have reduced the incidence of these handicaps, untoward side effects abound. Using a murine model of sublethal hypoxia, we demonstrated reduction in several transcription factors that modulate expression of genes known to be involved in several neural functions. We demonstrate the induction of these genes by minocycline, a tetracycline antibiotic with noncanonical functions, in both in vitro and in vivo studies. Specifically, there was induction of genes, including Sox10, Hif1a, Hif2a, Birc5, Yap1, Epo, Bdnf, Notch1 (cleaved), Pcna, Mag, Mobp, Plp1, synapsin, Adgra2, Pecam1, and reduction in activation of caspase 3, all known to affect proliferation, apoptosis, synaptic transmission, and nerve transmission. Minocycline treatment of mouse pups reared under sublethal hypoxic conditions resulted in improvement in open field testing parameters. These studies demonstrate beneficial effects of minocycline treatment following hypoxic insult, document up-regulation of several genes associated with improved cognitive function, and support the possibility of minocycline as a potential therapeutic target in the treatment of neurodevelopmental handicaps observed in the very premature newborn population. Additionally, these studies may aid in further interpretation of the effects of minocycline in the treatment trials and animal model studies of fragile X syndrome and multiple sclerosis.

A hydrogel-endothelial cell implant mimics infantile hemangioma: modulation by survivin and the Hippo pathway.

Microvascular endothelial cells cultured in three-dimensional hydrogel scaffolds form a network of microvessel structures when implanted subcutaneously in mice, inosculate with host vessels, and over time remodel into large ectatic vascular structures resembling hemangiomas. When compared with infantile hemangiomas, similarities were noted, including a temporal progression from a morphological appearance of a proliferative phase to the appearance of an involuted phase, mimicking the proliferative and involutional phases of infantile hemangioma. Consistent with the progression of a proliferative phase to an involuted phase, both the murine implants and human biopsy tissue exhibit reduced expression of Ajuba, YAP, and Survivin labeling as they progressed over time. Significant numbers of CD45+, CD11b+, Mac3+ mononuclear cells were found at the 2-week time point in our implant model that correlated with the presence of CD45+, CD68+ mononuclear cells observed in biopsies of human proliferative-phase hemangiomas. At the 4-week time point in our implant model, only small numbers of CD45+ cells were detected, which again correlated with our findings of significantly diminished CD45+, CD68+ mononuclear cells in human involutional-phase hemangiomas. The demonstration of mononuclear cell infiltration transiently in the proliferative phase of these lesions suggests that the vascular proliferation and/or regression may be driven in part by an immune response. Gross and microscopic morphological appearances of human proliferative and involutional hemangiomas and our implant model correlate well with each other as do the expression levels of Hippo pathway components (Ajuba and YAP) and Survivin and correlate with proliferation in these entities. Inhibitors of Survivin and Ajuba (which we have demonstrated to inhibit proliferation and increase apoptosis in murine hemangioendothelioma cell tissue culture) may have potential as other beneficial treatments for proliferating infantile hemangiomas. This implant model may have potential as a modest through-put screen for testing and development of therapeutics targeted at the proliferative phase of infantile hemangiomas, reducing the subsequent postinvolutional scarring or deformities sometimes associated with these lesions.

Disturbed shear stress reduces Klf2 expression in arterial-venous fistulae in vivo.

Laminar shear stress (SS) induces an antiproliferative and anti-inflammatory endothelial phenotype and increases Klf2 expression. We altered the diameter of an arteriovenous fistula (AVF) in the mouse model to determine whether increased fistula diameter produces disturbed SS in vivo and if acutely increased disturbed SS results in decreased Klf2 expression. The mouse aortocaval fistula model was performed with 22, 25, or 28 gauge needles to puncture the aorta and the inferior vena cava. Duplex ultrasound was used to examine the AVF and its arterial inflow and venous outflow, and SS was calculated. Arterial samples were examined with western blot, immunohistochemistry, and immunofluorescence analysis for proteins and qPCR for RNA. Mice with larger diameter fistulae had diminished survival but increased AVF patency. Increased SS magnitudes and range of frequencies were directly proportional to the needle diameter in the arterial limb proximal to the fistula but not in the venous limb distal to the fistula, with 22-gauge needles producing the most disturbed SS in vivo. Klf2 mRNA and protein expression was diminished in the artery proximal to the fistula in proportion to increasing SS. Increased fistula diameter produces increased SS magnitude and frequency, consistent with disturbed SS in vivo. Disturbed SS is associated with decreased mRNA and protein expression of Klf2. Disturbed SS and reduced Klf2 expression near the fistula are potential therapeutic targets to improve AVF maturation.

Temporal regulation of venous extracellular matrix components during arteriovenous fistula maturation.

PURPOSE: The venous limb of arteriovenous fistulae (AVF) adapts to the arterial environment by dilation and wall thickening; however, the temporal regulation of the expression of extracellular matrix (ECM) components in the venous limb of the maturing AVF has not been well characterized. We used a murine model of AVF maturation that recapitulates human AVF maturation to determine the temporal pattern of expression of these ECM components. METHODS: Aortocaval fistulae were created in C57BL/6J mice and the venous limb was analyzed on postoperative days 1, 3, 7, 21, and 42. A gene microarray analysis was performed on day 7; results were confirmed by qPCR, histology, and immunohistochemistry. Proteases, protease inhibitors, collagens, glycoproteins, and other non-collagenous proteins were characterized. RESULTS: The maturing AVF has increased expression of many ECM components, including increased collagen and elastin. Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase 1 (TIMP1) showed increased mRNA and protein expression during the first 7 days of maturation. Increased collagen and elastin expression was also significant at day 7. Expression of structural proteins was increased later during AVF maturation. Osteopontin (OPN) expression was increased at day 1 and sustained during AVF maturation. CONCLUSIONS: During AVF maturation, there is significantly increased expression of ECM components, each of which shows distinct temporal patterns during AVF maturation. Increased expression of regulatory proteins such as MMP and TIMP precedes increased expression of structural proteins such as collagen and elastin, potentially mediating a controlled pattern of ECM degradation and vessel remodeling without structural failure.

Adhesion molecule-mediated hippo pathway modulates hemangioendothelioma cell behavior.

Hemangioendotheliomas are categorized as intermediate-grade vascular tumors that are commonly localized in the lungs and livers. The regulation of this tumor cell's proliferative and apoptotic mechanisms is ill defined. We recently documented an important role for Hippo pathway signaling via endothelial cell adhesion molecules in brain microvascular endothelial cell proliferation and apoptosis. We found that endothelial cells lacking cell adhesion molecules escaped from contact inhibition and exhibited abnormal proliferation and apoptosis. Here we report on the roles of adherens junction molecule modulation of survivin and the Hippo pathway in the proliferation and apoptosis of a murine hemangioendothelioma (EOMA) cell. We demonstrated reduced adherens junction molecule (CD31 and VE-cadherin) expression, increased survivin and Ajuba expression, and a reduction in Hippo pathway signaling resulting in increased proliferation and decreased activation of effector caspase 3 in postconfluent EOMA cell cultures. Furthermore, we confirmed that YM155, an antisurvivin drug that interferes with Sp1-survivin promoter interactions, and survivin small interference RNA (siRNA) transfection elicited induction of VE-cadherin, decreased Ajuba expression, increased Hippo pathway and caspase activation and apoptosis, and decreased cell proliferation. These findings support the importance of the Hippo pathway in hemangioendothelioma cell proliferation and survival and YM155 as a potential therapeutic agent in this category of vascular tumors.

NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia.

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966-15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.

CD44 regulation of endothelial cell proliferation and apoptosis via modulation of CD31 and VE-cadherin expression.

CD44 has been implicated in a diverse array of cell behaviors and in a diverse range of signaling pathway activations under physiological and pathophysiological conditions. We have documented a role for CD44 in mediating vascular barrier integrity via regulation of PECAM-1 (CD31) expression. We now report our findings on the roles of CD44 in modulating proliferation and apoptosis of microvascular endothelial cells via its modulation of CD31 and VE-cadherin expression and the Hippo pathway. In this report, we demonstrate persistent increased proliferation and reduced activations of both effector and initiator caspases in high cell density, postconfluent CD44 knock-out (CD44KO), and CD31KO cultures. We found that reconstitution with murine CD44 or CD31 restored the proliferative and caspase activation rates to WT levels. Moreover, we have confirmed that the CD31 ecto-domain plays a key role in specific caspase cascades as well as cell adhesion-mediated cell growth and found that CD31 deficiency results in a reduction in VE-cadherin expression. Last, we have shown that both CD44KO and CD31KO endothelial cells exhibit a reduced VE-cadherin expression correlating with increased survivin expression and YAP nuclear localization, consistent with inactivation of the Hippo pathway, resulting in increased proliferation and decreased apoptosis. These findings support the concept that CD44 mediates several of its effects on endothelia through modulation of adhesion protein expression, which, in addition to its known modulation of junctional integrity, matrix metalloproteinase levels and activation, interactions with cortical membrane proteins, and selected signaling pathways, plays a key role as a critical regulator of vascular function.

Stratified control of IGF-I expression by hypoxia and stress hormones in osteoblasts.

Bone cells respond to the integrated effects of local and systemic regulation. Here we show that hypoxia and the stress hormones PGE2 and glucocorticoid interact in complex ways in osteoblasts, converging on insulin like growth factor I (IGF-I) expression. Whereas hypoxia alone rapidly increased transcription factor HIF activity, it suppressed DNA synthesis, had no significant effects on protein synthesis or alkaline phosphatase activity, and drove discrete changes in a panel of osteoblast mRNAs. Notably, hypoxia increased expression of the acute phase response transcription factor C/EBPδ which can induce IGF-I in response to PGE2, but conversely prevented the stimulatory effect of PGE2 on IGF-I mRNA. However, unlike its effect on C/EBPδ, hypoxia suppressed expression of the obligate osteoblast transcription factor Runx2, which can activate an upstream response element in the IGF-I gene promoter. Hypoxic inhibition of IGF-I and Runx2 were enforced by glucocorticoid, and continued with prolonged exposure. Our studies thus reveal that IGF-I expression is stratified by two critical transcriptional elements in osteoblasts, which are resolved by the individual and combined effects of hypoxic stress and stress hormones. In so doing, hypoxia suppresses Runx2, limits the enhancing influence of PGE2, and interacts with glucocorticoid to reduce IGF-I expression by osteoblasts.