GSAO-based molecular targeting of myocardial necrosis in acute ischemic insult

Purpose: GSAO has an arsenic group that binds to dithiols on various intracellular proteins that are upregulated during cellular stress including HSP90, PDI, which plays a role in the unfolded protein response and apoptosis, and Beclin-1, which drives autophagy. Extracellular dithiols are uncommon. As 111In-GSAO is membrane impermeable, intracellular uptake shows membrane disruption, a hallmark of necrotic cell death. It is proposed that information about preferential protein binding other than HSP90 would determine the likely initial cell death pathway. The current study sought to show feasibility of GSAO imaging in animal models of acute and chronic myocardial infarction and to evaluate the relation between myocardial apoptosis and necrosis. Methods: Chronic and acute myocardial infarction were induced by persistent occlusion and by 30 minutes occlusion + release of the LAD in rabbits and mice. In acute MI rabbits, in vivo and ex vivo SPECT imaging using GSAO (n = 6), GSAO + sestamibi (n = 4), GSAO + AA5 (n = 3) and control compound GSCA (n = 5) were performed, followed by gamma-counting and immunohistochemistry. In mice, ex vivo imaging and gamma-counting using GSAO was performed after acute MI (n = 6), after chronic MI at 2 (n = 6), 4 (n = 6) and 12 (n = 8) weeks and in controls (n = 5). GSCA was employed at 2 (n = 6) weeks. Six mice received fluorescently labeled AA5 and GSAO after acute MI. Results: In rabbits, myocardial GSAO uptake was high (1.1 ± 0.43%ID/g), localized in the perfusion defect and was significantly higher than in the remote area (0.07 ± 0.03%ID/g,

Inhibition of Neutral Sphingomyelinase 2 by Novel Small Molecule Inhibitors Results in Decreased Release of Extracellular Vesicles by Vascular Smooth Muscle Cells and Attenuated Calcification

Vascular calcification (VC) is an important contributor and prognostic factor in the pathogenesis of cardiovascular diseases. VC is an active process mediated by the release of extracellular vesicles by vascular smooth muscle cells (VSMCs), and the enzyme neutral sphingomyelinase 2 (nSMase2 or SMPD3) plays a key role. Upon activation, the enzyme catalyzes the hydrolysis of sphingomyelin, thereby generating ceramide and phosphocholine. This conversion mediates the release of exosomes, a type of extracellular vesicles (EVs), which ultimately forms the nidus for VC. nSMase2 therefore represents a drug target, the inhibition of which is thought to prevent or halt VC progression. In search of novel druglike small molecule inhibitors of nSMase2, we have used virtual ligand screening to identify potential ligands. From an in-silico collection of 48,6844 small druglike molecules, we selected 996 compounds after application of an in-house multi-step procedure combining different filtering and docking procedures. Selected compounds were functionally tested in vitro; from this, we identified 52 individual hit molecules that inhibited nSMase2 activity by more than 20% at a concentration of 150 mu M. Further analysis showed that five compounds presented with IC(50)s lower than 2 mu M. Of these, compounds ID 5728450 and ID 4011505 decreased human primary VSMC EV release and calcification in vitro. The hit molecules identified here represent new classes of nSMase2 inhibitors that may be developed into lead molecules for the therapeutic or prophylactic treatment of VC

Immunoglobulin G anti-endothelial cell antibodies: inducers of endothelial cell apoptosis in pulmonary arterial hypertension?

Endothelial cell (EC) apoptosis seems to play an important role in the pathophysiology of pulmonary arterial hypertension (PAH). We aimed to test the hypothesis that circulating anti-endothelial cell antibodies (AECA) of PAH patients induce EC apoptosis. Immunoglobulin (Ig)G was purified from sera of PAH patients (n = 26), patients with systemic lupus erythematosus (SLE) nephritis without PAH (n = 16), patients with systemic sclerosis (SSc) without PAH (n = 58) and healthy controls (n = 14). Human umbilical vein endothelial cells (HUVECs) were incubated with patient or healthy control IgG for 24 h. Thereafter, apoptosis was quantified by annexin A5 binding and hypoploid cell enumeration by flow cytometry. Furthermore, real-time cell electronic sensing (RT-CES) technology was used to monitor the effects of purified IgG from patient and healthy control IgG on HUVECs. As demonstrated previously, IgG of AECA-positive SLE nephritis patients (n = 7) induced a higher percentage of apoptosis of HUVECs compared to IgG of AECA-negative SLE nephritis patients and healthy controls. Furthermore, IgG of AECA-positive SLE nephritis patients induced a marked decrease in cell index as assessed by RT-CES technology. IgG of AECA-positive PAH patients (n = 12) and SSc patients (n = 13) did not alter the percentage of HUVEC apoptosis or cell index compared to IgG of AECA-negative PAH and SSc patients and healthy controls. AECA-positive PAH patients, in contrast to SLE nephritis patients, do not have circulating IgG AECA that enhances apoptosis of HUVECs in vitro. Further studies should focus on other mechanisms by which AECA may enhance EC apoptosis in PAH, such as antibody-dependent cell-mediated cytotoxicity