scholarly journals Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

2021 ◽  
Vol 4 (4) ◽  
pp. 74
Author(s):  
Molly Yao ◽  
Samayita Ganguly ◽  
Jane Hae Soo Shin ◽  
Tamer Elbayoumi

Homeostasis of vascular tone is intricately and delicately maintained systemically and locally, by autonomic nerves and hormones in the blood and by intimal vasoactive substances, respectively. The balance can be acutely or chronically interrupted secondary to many alterations, especially under pathological conditions. Excessive matricellular glycoprotein thrombospondin 1 (TSP1) levels in circulation have been found to play an important role in ischemia-reperfusion injuries of different organs, by acutely suppressing vasorelaxation and chronically remodeling vascular bed. Our laboratory has been interested in identifying new drug moieties, which can selectively and effectively counteract TSP1-induced vascular dysfunction, in order to address associated clinical complications. Preliminary studies using computational docking and molecular models revealed potential drug candidates for further evaluation via vascular functional bioassay to prove the antagonism using an ex vivo vascular model. Herein, we described an efficient screening method for the identification of active drug candidates, by adapting a multiwire myograph system to perform a protocol with different treatments, in the presence of pathological levels of TSP1. We discussed the promising pharmacological evaluation results and suggested suitable modification for versatile applications. We also described the necessity of pre-determination of optimal resting tension to obtain the maximal response, if the experimental test model is different from those with determined optimal resting tension.

2016 ◽  
Vol 21 (9) ◽  
pp. 956-964 ◽  
Author(s):  
Guruchandar Arulmozhivarman ◽  
Martin Stöter ◽  
Marc Bickle ◽  
Martin Kräter ◽  
Manja Wobus ◽  
...  

Hematopoietic stem and progenitor cells (HSPCs) generate all cell types of the blood and are crucial for homeostasis of all blood lineages in vertebrates. Hematopoietic stem cell transplantation (HSCT) is a rapidly evolving technique that offers potential cure for hematologic cancers, such as leukemia or lymphoma. HSCT may be autologous or allogenic. Successful HSCT depends critically on the abundance of engraftment-competent HSPCs, which are currently difficult to obtain in large numbers. Therefore, finding compounds that enhance either the number or the activity of HSPCs could improve prognosis for patients undergoing HSCT and is of great clinical interest. We developed a semiautomated screening method for whole zebrafish larvae using conventional liquid handling equipment and confocal microscopy. Applying this pipeline, we screened 550 compounds in triplicate for proliferation of HSPCs in vivo and identified several modulators of hematopoietic stem cell activity. One identified hit was valproic acid (VPA), which was further validated as a compound that expands and maintains the population of HSPCs isolated from human peripheral blood ex vivo. In summary, our in vivo zebrafish imaging screen identified several potential drug candidates with clinical relevance and could easily be further expanded to screen more compounds.


2021 ◽  
Vol 14 (10) ◽  
pp. 1028
Author(s):  
Sevil Korkmaz-Icöz ◽  
Belinda Ballikaya ◽  
Jasmin Soethoff ◽  
Patricia Kraft ◽  
Alex Ali Sayour ◽  
...  

Vascular ischemia/reperfusion injury (IRI) in patients undergoing coronary artery bypass grafting can result in graft failure and the need for repeat revascularization procedures. DuraGraft® has been shown to protect structure and function in saphenous vein grafts against IRI. We compared the effect of DuraGraft® to saline solution on arterial grafts submitted to IRI. Rat thoracic aortic rings were harvested and immediately mounted in organ bath chambers (control, n = 7 rats) or underwent cold ischemic preservation either in saline (IR, n = 9 rats) or DuraGraft® (IR+Dura, n = 9 rats). Vascular function was measured ex vivo and immunohistochemistry was performed. Impaired maximum vasorelaxation (Rmax) to ACh in the IR-group compared to controls was ameliorated by DuraGraft®, indicating an improvement in endothelial function (Rmax to ACh (%): IR + Dura 73 ± 2 vs. IR 48 ± 3, p < 0.05). Additionally, decreased aortic ring sensitivity to ACh (pD2-value: -log 50% maximum response) seen after IR in the saline group was increased by DuraGraft® (pD2 to ACh: IR+Dura 7.1 ± 0.1 vs. IR 6.3 ± 0.2, p < 0.05). Impaired maximum contractile response to phenylephrine and high potassium chloride concentrations in the IR group compared to controls was significantly improved by DuraGraft®. DuraGraft® alleviates vascular dysfunction following IRI by reducing nitro-oxidative stress and the expression of ICAM-1, without leukocytes engagement.


Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 642
Author(s):  
Molly Yao ◽  
Jalicia Sturdivant ◽  
Aren Ebrahimi ◽  
Samayita Ganguly ◽  
Tamer Elbayoumi

Elevated thrombospondin 1 (TSP1) is a prevalent factor, via cognate receptor CD47, in the pathogenesis of cardiovascular conditions, including ischemia-reperfusion injury (IRI) and pulmonary arterial hypertension (PAH). Moreover, TSP1/CD47 interaction has been found to be associated with platelet hyperaggregability and impaired nitric oxide response, exacerbating progression in IRI and PAH. Pathological TSP1 in circulation arises as a target of our novel therapeutic approach. Our “proof-of-concept” pharmacological strategy relies on recombinant human CD47 peptide (rh-CD47p) as a decoy receptor protein (DRP) to specifically bind TSP1 and neutralize TSP1-impaired vasorelaxation, strongly implicated in IRI and PAH. The binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western blotting and further quantified with modified ELISA, which also revealed a linear molar dose-dependent interaction. Ex vivo, pretreatment protocol with rh-CD47p (rh-CD47p added prior to TSP1 incubation) demonstrated a prophylactic effect against TSP1-impairment of endothelium-dependent vasodilation. Post-treatment set-up (TSP1 incubation prior to rh-CD47p addition), mimicking pre-existing excessive TSP1 in PAH, reversed TSP1-inhibited vasodilation back to control level. Dose titration identified an effective molar dose range (approx. ≥1:3 of tTSP1:rh-CD47p) for prevention of/recovery from TSP1-induced vascular dysfunction. Our results indicate the great potential for proposed novel decoy rh-CD47p-therapy to abrogate TSP1-associated cardiovascular complications, such as PAH.


2021 ◽  
Vol 22 (12) ◽  
pp. 6399
Author(s):  
Ioanna Papatheodorou ◽  
Eleftheria Galatou ◽  
Georgios-Dimitrios Panagiotidis ◽  
Táňa Ravingerová ◽  
Antigone Lazou

Accumulating evidence support the cardioprotective properties of the nuclear receptor peroxisome proliferator activated receptor β/δ (PPARβ/δ); however, the underlying mechanisms are not yet fully elucidated. The aim of the study was to further investigate the mechanisms underlying PPARβ/δ-mediated cardioprotection in the setting of myocardial ischemia/reperfusion (I/R). For this purpose, rats were treated with PPARβ/δ agonist GW0742 and/or antagonist GSK0660 in vivo and hearts were subjected to ex vivo global ischemia followed by reperfusion. PPARβ/δ activation improved left ventricular developed pressure recovery, reduced infarct size (IS) and incidence of reperfusion-induced ventricular arrhythmias while it also up-regulated superoxide dismutase 2, catalase and uncoupling protein 3 resulting in attenuation of oxidative stress as evidenced by the reduction in 4-hydroxy-2-nonenal protein adducts and protein carbonyl formation. PPARβ/δ activation also increased both mRNA expression and enzymatic activity of aldehyde dehydrogenase 2 (ALDH2); inhibition of ALDH2 abrogated the IS limiting effect of PPARβ/δ activation. Furthermore, upregulation of PGC-1α and isocitrate dehydrogenase 2 mRNA expression, increased citrate synthase activity as well as mitochondrial ATP content indicated improvement in mitochondrial content and energy production. These data provide new mechanistic insight into the cardioprotective properties of PPARβ/δ in I/R pointing to ALDH2 as a direct downstream target and suggesting that PPARβ/δ activation alleviates myocardial I/R injury through coordinated stimulation of the antioxidant defense of the heart and preservation of mitochondrial function.


2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Joshua Oluwasegun Bamidele ◽  
George Oche Ambrose ◽  
Oluwaseun Suleiman Alakanse

AbstractHSP90 is observed as one of the copious molecular chaperones that play a key role in mediating appropriate folding, maturation, and firmness of many client proteins in cells. The expression rate of HSP90 in cancer cells is at a level of 2- to 10-fold higher than the 1- to 2-fold of its unstressed and healthy ones. To combat this, several inhibitors to HSP90 protein have been studied (such as geldanamycin and its derivative 17-AAG and 17-DMAG) and have shown some primary side effects including plague, nausea, vomiting, and liver toxicity, hence the search for the best-in-class inhibitor for this protein through in silico. This study is aimed at analyzing the inhibitory potency of oxypeucedanin-a furocoumarin derivations, which have been reported to have antipoliferative activity in human prostrate carcinoma DN145 cells, and three other drug candidates retrieved from the literature via computational docking studies. The results showed oxypeucedanin as the compound with the highest binding energy of −9.2 kcal/mol. The molecular docking study was carried out using PyRx, Auto Dock Vina option, and the target was validated to confirm the proper target and the docking procedure employed for this study.


2021 ◽  
Vol 22 (10) ◽  
pp. 5172
Author(s):  
Thomas Prudhomme ◽  
John F. Mulvey ◽  
Liam A. J. Young ◽  
Benoit Mesnard ◽  
Maria Letizia Lo Faro ◽  
...  

Maintaining organ viability between donation and transplantation is of critical importance for optimal graft function and survival. To date in pancreas transplantation, static cold storage (SCS) is the most widely practiced method of organ preservation. The first experiments in ex vivo perfusion of the pancreas were performed at the beginning of the 20th century. These perfusions led to organ oedema, hemorrhage, and venous congestion after revascularization. Despite these early hurdles, a number of factors now favor the use of perfusion during preservation: the encouraging results of HMP in kidney transplantation, the development of new perfusion solutions, and the development of organ perfusion machines for the lung, heart, kidneys and liver. This has led to a resurgence of research in machine perfusion for whole organ pancreas preservation. This review highlights the ischemia-reperfusion injuries assessment during ex vivo pancreas perfusion, both for assessment in pre-clinical experimental models as well for future use in the clinic. We evaluated perfusion dynamics, oedema assessment, especially by impedance analysis and MRI, whole organ oxygen consumption, tissue oxygen tension, metabolite concentrations in tissue and perfusate, mitochondrial respiration, cell death, especially by histology, total cell free DNA, caspase activation, and exocrine and endocrine assessment.


2011 ◽  
Vol 300 (1) ◽  
pp. H144-H153 ◽  
Author(s):  
Alexis Ascah ◽  
Maya Khairallah ◽  
Frédéric Daussin ◽  
Céline Bourcier-Lucas ◽  
Richard Godin ◽  
...  

Susceptibility of cardiomyocytes to stress-induced damage has been implicated in the development of cardiomyopathy in Duchenne muscular dystrophy, a disease caused by the lack of the cytoskeletal protein dystrophin in which heart failure is frequent. However, the factors underlying the disease progression are unclear and treatments are limited. Here, we tested the hypothesis of a greater susceptibility to the opening of the mitochondrial permeability transition pore (PTP) in hearts from young dystrophic ( mdx) mice (before the development of overt cardiomyopathy) when subjected to a stress protocol and determined whether the prevention of a PTP opening is involved in the cardioprotective effect of sildenafil, which we have previously reported in mdx mice. Using the 2-deoxy-[3H]glucose method to quantify the PTP opening in ex vivo perfused hearts, we demonstrate that when compared with those of controls, the hearts from young mdx mice subjected to ischemia-reperfusion (I/R) display an excessive PTP opening as well as enhanced activation of cell death signaling, mitochondrial oxidative stress, cardiomyocyte damage, and poorer recovery of contractile function. Functional analyses in permeabilized cardiac fibers from nonischemic hearts revealed that in vitro mitochondria from mdx hearts display normal respiratory function and reactive oxygen species handling, but enhanced Ca2+ uptake velocity and premature opening of the PTP, which may predispose to I/R-induced injury. The administration of a single dose of sildenafil to mdx mice before I/R prevented excessive PTP opening and its downstream consequences and reduced tissue Ca2+ levels. Furthermore, mitochondrial Ca2+ uptake velocity was reduced following sildenafil treatment. In conclusion, beyond our documentation that an increased susceptibility to the opening of the mitochondrial PTP in the mdx heart occurs well before clinical signs of overt cardiomyopathy, our results demonstrate that sildenafil, which is already administered in other pediatric populations and is reported safe and well tolerated, provides efficient protection against this deleterious event, likely by reducing cellular Ca2+ loading and mitochondrial Ca2+ uptake.


2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Maria P McGee ◽  
Michael Morykwas ◽  
James Jordan ◽  
Louis Argenta

Interstitial edema is an early response to myocardial ischemia, leading to fibrosis and remodeling in several heart failure conditions. We aimed to clarify whether osmotic, frictional, or mechanical forces drive fluid accumulation. Equilibrium and dynamic interstitial hydration parameters were determined, compared, and analyzed using osmotic stress approaches in explants from ischemic and nonischemic myocardial regions of pig heart. They were isolated after injury induced by ligating 3-4 branches of the left anterior descending coronary artery, for 85 min followed by 3 hours’ reperfusion. Their volume changed (Δ V max ) linearly with colloidosmotic pressure in both ischemic and nonischemic areas, yielding interstitial compliance values of 1.04 ± 0.09 and 1.08 ± 0.05 µl/g/ mmHg , which do not differ significantly, and hydration potentials from the abscissa intercepts at Δ V max = 0, of -121.4 ± 28 and -14.7 ± 7.6 mmHg, which do (mean ± SE, n = 5 , P-value = 0.001). These hydration potential differences manifest ex-vivo influx rates 8.5 ± 2.7- fold slower in ischemic than nonischemic myocardium. Surprisingly, interstitial flow resistance values derived from net-flow rates at an imposed pressure difference of 216 mmHg were 0.23 ± 0.08 and 0.19 ± 0.01 µl -1 . g. min and did not differ significantly between the areas. The similarity in interstitial compliance and fluid resistance indicates that the more negative hydration potential and faster efflux rates in at-risk regions after reperfusion are due to increased hydrostatic pressure rather than decreased osmotic or frictional forces. Tissue distends due to interstitial fluid accumulation against matrix mechanical forces, including elastic recoil of the collagen elastin mesh and fibroblast action, consistent with impaired drainage and persistent diastolic-like conditions during reperfusion of at-risk areas in vivo . These results indicate changes in pressure gradient magnitude and may have clinical and therapeutic implications; for example, reversal of paracrine interstitial flows during early remodeling


Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Louisa Mezache ◽  
Heather Struckman ◽  
Anna Phillips ◽  
Stephen Baine ◽  
Amara Greer-short ◽  
...  

Atrial fibrillation (AF), the most common arrhythmia, is associated with inflammation and vascular dysfunction. AF patients have elevated levels of vascular endothelial growth factor (VEGF; 90-580 pg/ml), which promotes vascular leak and edema. We have previously identified edema-induced disruption of sodium channel (Na V 1.5) -rich intercalated disk (ID) nanodomains as a novel arrhythmia mechanism. We hypothesized that (i) elevated VEGF levels promote AF by disrupting ID nanodomains, and slowing atrial conduction, and (ii) protection of the vascular barrier can prevent these arrhythmias. Clinically-relevant VEGF levels (500 pg/ml, 60 minutes) increased FITC-dextran extravasation (99.3% vs. 24.3% in vehicle controls) in WT mouse hearts, consistent with increased vascular leak. Electron microscopy revealed ID nanodomain swelling, near both gap junctions (perinexi; 64±9nm vs 17±1nm) and mechanical junctions (63±4nm vs 27±2nm) in VEGF-treated hearts relative to controls. Super-resolution STORM microscopy revealed Na V 1.5 enrichment at perinexi (9±2 fold) and N-cadherin-rich sites (7±1 fold) relative to non-junctional ID sites in control hearts. VEGF reduced Na V 1.5 enrichment at both sites (6±1 and 4±1 fold, respectively), consistent with Na V 1.5 translocation from ID nanodomains. Atrial conduction, assessed by optical mapping, was slowed by VEGF (10±0.4 cm/s vs 21.3±1.3 cm/s at baseline). VEGF increased atrial arrhythmia burden both ex vivo (80% vs 0% in vehicle controls) and in vivo (70% vs 20% in vehicle controls). Next, we tested two strategies shown to prevent vascular barrier breakdown. Blocking connexin43 hemichannels (αCT11 peptide) decreased both incidence (40%) and duration (1.45±3.42s) of VEGF-induced arrhythmias. Likewise, blocking pannexin1 channels (Panx1-IL2 peptide) shortened VEGF-induced arrhythmias (2.48±0.83s). Mefloquine and spironolactone, which are small molecules that respectively inhibit Cx43 hemichannels and pannexin channels, were also found to effectively prevent VEGF-induced atrial arrhythmias. These results highlight VEGF-induced vascular leak as a novel mechanism for AF, and suggest vascular barrier protection as an anti-arrhythmic strategy.


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