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.
CD44 mediates vascular barrier integrity via regulation of VE-cadherin and CD31 expression.
CD44 regulates cell proliferation and specific caspase-mediated apoptosis via modulation of CD31 and VE-cadherin expression through the Hippo pathway.
CD44 modulates proliferation and apoptosis of microvascular endothelial cells.
CD44 regulates endothelial cell survival by modulating specific cell adhesion molecules via the Hippo pathway.
Display omitted
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.
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.
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 β-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.
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.
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.
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.
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.
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;
=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,
=0.0015; interleukin-10: 7.6-fold,
=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;
=0.0306), increased collagen density (2.4-fold;
=0.0432), and increased number of M2 macrophages (2.1-fold;
=0.0335).
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.
PECAM-1: old friend, new partners Ilan, Neta; Madri, Joseph A
Current opinion in cell biology,
10/2003, Letnik:
15, Številka:
5
Journal Article
Recenzirano
The maintenance of vascular function is of paramount importance to an organism’s existence. PECAM-1 (CD31), first thought of as a marker for endothelia, has been shown to be an important scaffolding ...molecule involved in several signaling pathways. Recent studies have demonstrated an even wider range of functions for this versatile molecule including participation in maintenance of adherens junction integrity and permeability, organization of the intermediate filament cytoskeleton, regulation of catenin localization and transcriptional activities, participation in STAT isoform signaling, control of apoptotic events, and modulation of cardiac cushion development.
Adhesion molecule CD44 is expressed by multiple cell types and is implicated in various cellular and immunological processes. In this study, we examined the effect of global CD44 deficiency on myelin ...oligodendrocyte glycoprotein peptide (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Compared to C57BL/6 wild-type mice, CD44-deficient mice presented with greater disease severity, increased immune cell numbers in the central nervous system, and increased anti-MOG antibody and proinflammatory cytokine production, especially those associated with T helper 17 (Th17) cells. Further, decreased numbers of peripheral CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) were observed in CD44-knockout mice throughout the disease course. CD44-knockout CD4 T cells exhibited reduced transforming growth factor-β receptor type I (TGF-β RI) expression that did not impart a defect in Treg polarization in vitro, but did correlate with enhanced Th17 polarization in vitro. Further, EAE in bone marrow-chimeric animals suggested CD44 expression on both circulating and noncirculating cells limited disease severity. Endothelial expression of CD44 limited T-cell adhesion to and transmigration through murine endothelial monolayers in vitro. Importantly, we also identified increased permeability of the blood-brain barrier in vivo in CD44-deficient mice before and following immunization. These data suggest that CD44 has multiple protective roles in EAE, with effects on cytokine production, T-cell differentiation, T-cell-endothelial cell interactions, and blood-brain barrier integrity.
PDF
