Sonia A. Cunningham

JAM2 interacts with alpha4beta1. Facilitation by JAM3.

We have previously reported that junctional adhesion molecule 2 (JAM2) adheres to T cells through heterotypic interactions with JAM3. An examination of the cation dependence of JAM2 adhesion to HSB cells revealed a Mn2+-enhanced binding component indicative of integrin involvement. Using neutralizing integrin antibodies, we have defined an interaction between JAM2 and α4β1 in T cells. The interaction is readily amenable to drug intervention as demonstrated by the ability of TBC 772, an α4-specific inhibitor, to attenuate the Mn2+-enhanced component. Intriguingly, the engagement of α4β1 by JAM2 is only enabled following prior adhesion of JAM2 with JAM3 and is not detectable in cells where JAM3 expression is absent. Supporting this observation, we show that neutralizing JAM3 serum and soluble JAM3 ectodomain inhibit not only JAM2 binding to JAM3 but also prevent JAM2/α4β1 interactions in T cells. We further define the first Ig-like fold of JAM2 as being competent in binding both JAM3 and α4β1counter-receptors. Mutagenesis of the only acidic residue in the C-D loop of this Ig fold, namely Asp-82, has no bearing on α4β1 interactions, and thus JAM2 deviates somewhat from the mechanism used by other immunoglobulin superfamily cell adhesion molecules to engage integrin.

Characterization of Vascular Endothelial Cell Growth Factor Interactions with the Kinase Insert Domain-containing Receptor Tyrosine Kinase A REAL TIME KINETIC STUDY

The kinase insert domain-containing receptor (KDR) tyrosine kinase mediates calcium mobilization in endothelial cells and plays a key role during physiological and pathological angiogenesis. To provide a detailed understanding of how KDR is activated, we analyzed the kinetics of ligand-receptor interaction using BIAcore. Both predimerized (KDR-Fc) and monomeric (KDR-cbu) receptors were examined with vascular endothelial cell growth factor (VEGF) homodimers and VEGF/placental growth factor (PlGF) heterodimers. VEGF binds to KDR-Fc with ka = 3.6 ± 0.07e6, kd = 1.34 ± 0.19e−4, and KD = 37.1 ± 4.9 pm. These values are similar to those displayed by monomeric KDR where ka = 5.23 ± 1.4e6, kd = 2.74 ± 0.76e−4, and KD = 51.7 ± 5.8 pm were apparent. In contrast, VEGF/PlGF bound to KDR-Fc with ka = 7.3 ± 1.6e4,kd = 4.4 ± 1.2e−4, andKD = 6 ± 1.2 nm. Thus, the heterodimer displays a 160-fold reduced KD for binding to predimerized KDR, which is mainly a consequence of a 50-fold reduction in ka . We were unable to detect association between VEGF/PlGF and monomeric KDR. However, nanomolar concentrations of VEGF/PlGF were able to elicit weak calcium mobilization in endothelial cells. This latter observation may indicate partial predimerization of KDR on the cell surface or facilitation of binding due to accessory receptors.

KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells.

We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR antibody is able to completely block VEGF165-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF165. Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca2+ in HUVEC in response to VEGF165.We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR antibody is able to completely block VEGF165-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF165. Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca2+ in HUVEC in response to VEGF165.

CFTR targeting in epithelial cells.

We used polarized and nonpolarized colonic cell lines (HT-29) to correlate CFTR function and expression with epithelial cell morphogenesis. Unpolarized cells express levels of CFTR mRNA and protein that are equivalent to those observed in polarized cells, and the extent of CFTR glycosylation is also similar. Despite these similarities in CFTR expression, the polarized cells secreted Cl in response tocAMP, but there was nocAMP-stimulated Cl conductance response in the unpolarized cells. In the polarized cells, CFTR is localized in the apical membrane domain, but in unpolarized cells the protein is retained at a perinuclear location. These findings indicate that a peripheral targeting mechanism, distal to the Golgi cisternae, controls the progression of N-linked glycoproteins like CFTR to the apical membrane. This targeting process does not become active until epithelial cells polarize. It may determine whether mutant forms of CFTR are targeted to the apical membrane.

Stratification of foot ulcer risk in patients with diabetes: a population-based study: Stratification of Foot Ulcer Risk

This trial assessed whether a simple clinical tool can be used to stratify patients with diabetes, according to risk of developing foot ulceration. This was a prospective, observational follow‐up study of 3526 patients with diabetes (91% type 2 diabetes) attending for routine diabetes care. Mean age was 64.7 (range 15–101) years and duration of diabetes was 8.8 (±1.5 SD) years. Patients were categorised into ‘low’ (64%), ‘moderate’ (23%) or ‘high’ (13%) risk of developing foot ulcers by trained staff using five clinical criteria during routine patient care. During follow‐up (1.7 years), 166 (4.7%) patients developed an ulcer. Foot ulceration was 83 times more common in high risk and six times more in moderate risk, compared with low‐risk patients. The negative predictive value of a ‘low‐risk score’ was 99.6% (99.5–99.7%; 95% confidence interval). This clinical tool accurately predicted foot ulceration in routine practice and could be used direct scarce podiatry resources towards those at greatest need.

Stratification of foot ulcer risk in patients with diabetes

This trial assessed whether a simple clinical tool can be used to stratify patients with diabetes, according to risk of developing foot ulceration. This was a prospective, observational follow‐up study of 3526 patients with diabetes (91% type 2 diabetes) attending for routine diabetes care. Mean age was 64.7 (range 15–101) years and duration of diabetes was 8.8 (±1.5 SD) years. Patients were categorised into ‘low’ (64%), ‘moderate’ (23%) or ‘high’ (13%) risk of developing foot ulcers by trained staff using five clinical criteria during routine patient care. During follow‐up (1.7 years), 166 (4.7%) patients developed an ulcer. Foot ulceration was 83 times more common in high risk and six times more in moderate risk, compared with low‐risk patients. The negative predictive value of a ‘low‐risk score’ was 99.6% (99.5–99.7%; 95% confidence interval). This clinical tool accurately predicted foot ulceration in routine practice and could be used direct scarce podiatry resources towards those at greatest need.

Protein Targeting and the Control Of C1− Secretion in Colonic Epithelial Cells

Human colonic epithelial cells can secrete chloride in response to cAMP-dependent stimulation following cellular polarization. This is correlated with the presence of 8 pS cAMP-activated Cl- channels within the apical membranes of these cells and their absence from the plasma membranes of unpolarized cells (Morris et al., 1992). The protein responsible for this cAMP-activated Cl-conductance is now recognized to be the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, Rommens et al., 1989; Bear et al., 1992). We have, therefore, postulated that the apical membrane expression of CFTR, and the ability of these cells to secrete Cl- in response to cAMP, is manifest when this protein targets to the apical membrane during epithelial cell differentiation (Morris et al., 1992).