A non-canonical role for desmoglein-2 in endothelial cells: implications for neoangiogenesis.

Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by non-desmosome-forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34(+)CD45(dim) hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34(+)CD45(dim)DSG2(+) progenitor cells are multi-potent and pro-angiogenic in vitro. Using a 'knockout-first' approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2 (lo/lo)) and observed that, in response to reduced levels of Dsg2: (i) CD31(+) ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrow-derived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell-cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.

Cardioprotective 3',4'-dihydroxyflavonol attenuation of JNK and p38(MAPK) signalling involves CaMKII inhibition.

DiOHF (3',4'-dihydroxyflavonol) is cardioprotective against I/R (ischaemia/reperfusion) injury. The biological activities of flavonols are associated with kinase modulation to alter cell signalling. We thus investigated the effects of DiOHF on the activation of MAPKs (mitogen-activated protein kinases) that regulate the cardiac stress response. In an ovine model of I/R, JNK (c-Jun N-terminal kinase), p38(MAPK), ERK (extracellular-signal-regulated kinase) and Akt were activated, and NP202, a pro-drug of DiOHF, reduced infarct size and inhibited JNK and p38(MAPK) activation, whereas ERK and Akt phosphorylation were unaltered. Similarly, in cultured myoblasts, DiOHF pre-treatment preserved viability and inhibited activation of JNK and p38(MAPK), but not ERK in response to acute oxidative and chemotoxic stress. Furthermore, DiOHF prevented stress-activation of the direct upstream regulators MKK4/7 (MAPK kinase 4/7) and MKK3/6 respectively. We utilized small-molecule affinity purification and identified CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) as a kinase targeted by DiOHF and demonstrated potent CaMKII inhibition by DiOHF in vitro. Moreover, the specific inhibition of CaMKII with KN-93, but not KN-92, prevented oxidative stress-induced activation of JNK and p38(MAPK). The present study indicates DiOHF inhibition of CaMKII and attenuation of MKK3/6→p38(MAPK) and MKK4/7→JNK signalling as a requirement for the protective effects of DiOHF against stress stimuli and myocardial I/R injury.

Protection against reperfusion injury by 3',4'-dihydroxyflavonol in rat isolated hearts involves inhibition of phospholamban and JNK2.

BACKGROUND: Flavonols, including 3',4'-dihydroxyflavonol (DiOHF), reduce myocardial ischemia and reperfusion (I/R) injury but their mechanism remains uncertain. To better understand the mechanism of the cardioprotective actions of flavonols we investigated the effect of DiOHF on cardiac function and the activation of protective and injurious signalling kinases after I/R in rat isolated hearts. METHODS: We assessed the effect of global ischemia (20min) and reperfusion (5-30min) on cardiac function and injury in rat isolated, perfused hearts in the absence or presence of DiOHF (10µM) during reperfusion. Western blotting was used to assess changes in the phosphorylation state of kinases known to be involved in injury or protection. RESULTS: DiOHF improved cardiac contractility and reduced perfusion pressure and cell death in the isolated hearts. Phosphorylation of p38MAPK and CaMKII increased during ischemia with no further increase during reperfusion. Phosphorylation of other kinases increased during reperfusion. Phosphorylation of phospholamban (PLN) peaked at 5min of reperfusion whereas phosphorylation of Akt, Erk, STAT3 and JNK2 was highest after 30min. The presence of DiOHF during reperfusion significantly inhibited the activation of PLN and JNK without affecting phosphorylation of the protective kinases Erk1/2 and STAT3. Experiments in vitro demonstrated that DiOHF inhibited CaMKII by competing with ATP but not Ca2+/calmodulin. CONCLUSIONS: It is proposed that DiOHF confers protection against myocardial reperfusion injury by inhibiting CaMKII and subsequent PLN-induced leak of Ca2+ from the sarcoplasmic reticulum as well as by inhibiting JNK2 activation to reduce apoptosis.