Brian G. Coon

Vascular remodeling is governed by a VEGFR3-dependent fluid shear stress set point

Vascular remodeling under conditions of growth or exercise, or during recovery from arterial restriction or blockage is essential for health, but mechanisms are poorly understood. It has been proposed that endothelial cells have a preferred level of fluid shear stress, or ‘set point’, that determines remodeling. We show that human umbilical vein endothelial cells respond optimally within a range of fluid shear stress that approximate physiological shear. Lymphatic endothelial cells, which experience much lower flow in vivo, show similar effects but at lower value of shear stress. VEGFR3 levels, a component of a junctional mechanosensory complex, mediate these differences. Experiments in mice and zebrafish demonstrate that changing levels of VEGFR3/Flt4 modulates aortic lumen diameter consistent with flow-dependent remodeling. These data provide direct evidence for a fluid shear stress set point, identify a mechanism for varying the set point, and demonstrate its relevance to vessel remodeling in vivo. DOI: http://dx.doi.org/10.7554/eLife.04645.001

Endothelial Nuclear Factor-κB–Dependent Regulation of Arteriogenesis and Branching

Background— Arteriogenesis and collateral formation are complex processes requiring integration of multiple inputs to coordinate vessel branching, growth, maturation, and network size. Factors regulating these processes have not been determined. Methods and Results— We used an inhibitor of NF&kgr;B activation (I&kgr;B&agr;SR) under control of an endothelial-specific inducible promoter to selectively suppress endothelial nuclear factor-&kgr;B activation during development, in the adult vasculature, or in vitro. Inhibition of nuclear factor-&kgr;B activation resulted in formation of an excessively branched arterial network that was composed of immature vessels and provided poor distal tissue perfusion. Molecular analysis demonstrated reduced adhesion molecule expression leading to decreased monocyte influx, reduced hypoxia-inducible factor-1&agr; levels, and a marked decrease in &dgr;-like ligand 4 expression with a consequent decrease in Notch signaling. The latter was the principal cause of increased vascular branching as treatment with Jagged-1 peptide reduced the size of the arterial network to baseline levels. Conclusions— These findings identify nuclear factor-&kgr;B as a key regulator of adult and developmental arteriogenesis and collateral formation. Nuclear factor-&kgr;B achieves this by regulating hypoxia-inducible factor-1&agr;–dependent expression of vascular endothelial growth factor-A and platelet-derived growth factor-BB, which are necessary for the development and maturation of the arterial collateral network, and by regulating &dgr;-like ligand 4 expression, which in turn determines the size and complexity of the network.

Lowe syndrome patient fibroblasts display Ocrl1-specific cell migration defects that cannot be rescued by the homologous Inpp5b phosphatase

Abstract The Lowe syndrome (LS) is a life-threatening, developmental disease characterized by mental retardation, cataracts and renal failure. Although this human illness has been linked to defective function of the phosphatidylinositol 5-phosphatase, Ocrl1 (Oculo-Cerebro-Renal syndrome ofLowe protein1), the mechanism by which this enzyme deficiency triggers the disease is not clear. Ocrl1 is known to localize mainly to the Golgi apparatus and endosomes, however it translocates to plasma membrane ruffles upon cell stimulation with growth factors. The functional implications of this inducible translocation to the plasma membrane are presently unknown. Here we show that Ocrl1 is required for proper cell migration, spreading and fluid-phase uptake in both established cell lines and human dermal fibroblasts. We found that primary fibroblasts from two patients diagnosed with LS displayed defects in these cellular processes. Importantly, these abnormalities were suppressed by expressing wild-type Ocrl1 but not by a phosphatase-deficient mutant. Interestingly, the homologous human PI-5-phosphatase, Inpp5b, was unable to complement the Ocrl1-dependent cell migration defect. Further, Ocrl1 variants that cannot bind the endocytic adaptor AP2 or clathrin, like Inpp5b, were less apt to rescue the migration phenotype. However, no defect in membrane recruitment of AP2/clathrin or in transferrin endocytosis by patient cells was detected. Collectively, our results suggest that Ocrl1, but not Inpp5b, is involved in ruffle-mediated membrane remodeling. Our results provide new elements for understanding how Ocrl1 deficiency leads to the abnormalities associated with the LS.

JNK2 Promotes Endothelial Cell Alignment under Flow

Endothelial cells in straight, unbranched segments of arteries elongate and align in the direction of flow, a feature which is highly correlated with reduced atherosclerosis in these regions. The mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) is activated by flow and is linked to inflammatory gene expression and apoptosis. We previously showed that JNK activation by flow is mediated by integrins and is observed in cells plated on fibronectin but not on collagen or basement membrane proteins. We now show thatJNK2 activation in response to laminar shear stress is biphasic, with an early peak and a later peak. Activated JNK localizes to focal adhesions at the ends of actin stress fibers, correlates with integrin activation and requires integrin binding to the extracellular matrix. Reducing JNK2 activation by siRNA inhibits alignment in response to shear stress. Cells on collagen, where JNK activity is low, align slowly. These data show that an inflammatory pathway facilitates adaptation to laminar flow, thereby revealing an unexpected connection between adaptation and inflammatory pathways.

The Epsin Family of Endocytic Adaptors Promotes Fibrosarcoma Migration and Invasion

Abnormalities in the process of endocytosis are classically linked to malignant transformation through the deficient down-regulation of signaling receptors. The present study describes a non-classical mechanism that does not require internalization by which endocytic proteins affect cell migration and basement membrane invasion. Specifically, we found that the endocytic adaptor epsin binds and regulates the biological properties of the signaling molecule RalBP1 (Ral-binding protein 1). Epsin interacted with the N terminus of RalBP1 via its characteristic epsin N-terminal homology (ENTH) domain. A combination of siRNA-mediated knock-down and transfection of siRNA-resistant constructs in fibrosarcoma cells demonstrated that impairment of the epsin-RalBP1 interaction led to cell migration and basement membrane invasion defects. We found the ENTH domain was necessary and sufficient to sustain normal cell migration and invasion. Because all the epsin endocytic motifs reside in the C-terminal part of the molecule, these results suggest that this novel regulatory circuit does not require endocytosis. In addition, cells depleted of epsin-RalBP1 complex displayed deficient activation of Rac1 and Arf6 suggesting a signaling function for this novel interaction. Further, overexpression of either epsin or RalBP1 enhanced migration and invasion of fibrosarcoma cells. Collectively, our results indicate that epsin regulates RalBP1 function in Rac1- and Arf6-dependent pathways to ultimately affect cell migration and invasion. We propose that the observed up-regulation of both epsin and RalBP1 in certain cancers contributes to their invasive characteristics.

The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway

The epsins are a family of adaptors involved in recruiting other endocytic proteins, binding of ubiquitylated cargo and induction of membrane curvature. These molecules bear a characteristic epsin N-terminal homology (ENTH) domain and multiple peptide motifs that mediate protein-protein interactions. We have previously demonstrated that the ENTH domain of epsin is involved in Cdc42 signaling regulation. Here, we present evidence that yeast epsin 2 (Ent2) plays a signaling role during cell division. We observed that overexpression of the ENTH domain of Ent2 (ENTH2), but not Ent1, promoted the formation of chains of cells and aberrant septa. This dominant-negative effect resulted from ENTH2-mediated interference with septin assembly pathways. We mapped the ENTH2 determinants responsible for induction of the phenotype and found them to be important for efficient binding to the septin regulatory protein, Bem3. Supporting a physiological role for epsin 2 in cell division, the protein localized to sites of polarized growth and cytokinesis and rescued a defect in cell division induced by Bem3 misregulation. Collectively, our findings provide a potential molecular mechanism linking endocytosis (via epsin 2) with signaling pathways regulating cell division.

Fibronectin attachment protein from bacillus Calmette-Guerin as targeting agent for bladder tumor cells

The adjuvant therapy of choice for superficial bladder cancer is the intravesical instillation of live Mycobacterium bovis bacillus Calmette‐Guerin (BCG). Despite the fact that this therapy is the most effective treatment for superficial bladder cancer, intravesical administration of BCG is associated with high local morbidity and the potential for systemic infection. Therefore, there is a need for the development of safer, less toxic approaches to fight this disease. Because fibronectin attachment protein (FAP) is a key element in BCG retention and targeting to cells, we hypothesize that this protein can be used as targeting agent to deliver cytotoxic cargo for the treatment of bladder tumors. Here, we evaluated the ability of bladder tumor cells to bind and endocytose FAP via fibronectin–integrin complexes. We found that microaggregation induced by an anti‐FAP polyclonal antibody accelerated FAP uptake by T24 bladder tumor cells. FAP was determined to be internalized via a clathrin‐independent, caveolae‐dependent mechanism. Furthermore, once within the endosomal compartment, FAP was targeted to the lysosomal compartment with negligible recycling to the plasma membrane. Importantly, we demonstrated that FAP microaggregation and internalization could also be triggered by multivalent Ni2+NTA‐bearing liposomes. Overall, our studies validate the use of FAP as a targeting vector and provide the foundation for the design of more effective, less‐toxic bladder cancer therapeutics.

Epsins’ novel role in cancer cell invasion

The epsin family of endocytic adaptors has been found to be upregulated in cancer; however the relevance of these findings to this pathological condition is unclear. We have recently demonstrated that epsins are required for cell migration. In fact, epsin overexpression promotes cancer cell invasion. Further, and in agreement with our previous findings, we also observed that overexpression of epsins led to epithelial cell migration beyond colony boundaries. Additionally, our results show that epsin-3 is the most potent paralog enhancing cell migration and invasion. Interestingly, epsin-3 expression is not widespread but highly restricted to migratory keratinocytes and aggressive carcinomas. Upon further investigation, we also identified epsin-3 as being expressed in pancreatic cancer cells. These findings suggest that upregulation of the EPN3 gene is specifically associated with invasive, aggressive cancers. We predict that investigation of these links between the endocytic machinery and mechanisms involved in tumor dissemination will contribute to the development of novel anti-metastatic and anti-cancer strategies.

Syndecan 4 controls lymphatic vasculature remodeling during mouse embryonic development

The role of fluid shear stress in vasculature development and remodeling is well appreciated. However, the mechanisms regulating these effects remain elusive. We show that abnormal flow sensing in lymphatic endothelial cells (LECs) caused by Sdc4 or Pecam1 deletion in mice results in impaired lymphatic vessel remodeling, including abnormal valve morphogenesis. Ablation of either gene leads to the formation of irregular, enlarged and excessively branched lymphatic vessels. In both cases, lymphatic valve-forming endothelial cells are randomly oriented, resulting in the formation of abnormal valves. These abnormalities are much more pronounced in Sdc4−/−; Pecam1−/− double-knockout mice, which develop severe edema. In vitro, SDC4 knockdown human LECs fail to align under flow and exhibit high expression of the planar cell polarity protein VANGL2. Reducing VANGL2 levels in SDC4 knockdown LECs restores their alignment under flow, while VANGL2 overexpression in wild-type LECs mimics the flow alignment abnormalities seen in SDC4 knockdown LECs. SDC4 thus controls flow-induced LEC polarization via regulation of VANGL2 expression. Highlighted article: Perturbation of syndecan 4 in mouse embryos reveals a new mechanism of fluid shear stress-mediated signaling involving syndecan 4-dependent upregulation of the PCP protein Vangl2.

Erratum: Mutations in the Histone Modifier PRDM6 Are Associated with Isolated Nonsyndromic Patent Ductus Arteriosus (American Journal of Human Genetics (2016) 98(6) (1082–1091) (S000292971630057X) (10.1016/j.ajhg.2016.03.022))

‘‘Dorothy unstintingly gave her time and energy to assist others. She volunteered for numerous activities to benefit the clinical genetics community. She was a founding fellow of the American College of Medical Genetics and also a member of the founding board of directors of the American Board of Medical Genetics, for which she served as treasurer and helped to make up queries for the first group of examinees for the board. Along with the other examiners, she had to take the next examination to become board certified and a fellow herself, a requirement she took with good humor.’’