osmotic-drug-delivery-to-ischemic-hindlimbs-perfusion-vasculature

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

The Texas Heart Institute at St. Luke's Episcopal Hospital

In innesti di vena safena autologa, prostaciclina (PGI(1)), una molecola vasoprotettiva prodotta dalle cellule endoteliali normali, &egrave; down-regolato rispetto all&#39;ingentilimento safene e le arterie carotide normale. Ridotta sintesi PGI(2) possono contribuire alla deposizione piastrinica locale, accumulo di cellule (VSMC) muscolatura liscia vascolare, l&#39;aterosclerosi e l&#39;insufficienza di innesti di bypass venoso. Abbiamo esaminato se gene trasferimento mediato iperespressione di COX-1 in vene innestati (1) aumenta PGI(2) e AMP ciclico (cAMP) produzione, (2) conduce al flusso di sangue locale in presenza di ipercolesterolemia e la vasodilatazione e (3) riduce la formazione di neointima.

Trasferimento del gene della COX-1 migliora la dimensione del lume e innesti di bypass del flusso sanguigno nella carotide.

Dans les greffes autologues de veine saph&egrave;ne, prostacycline (PGI(1)), une mol&eacute;cule vasoprotectrice produite par les cellules endoth&eacute;liales normales, est diminu&eacute;es par rapport &agrave; l&#39;ungrafted des veines saph&egrave;nes et des art&egrave;res carotides normales. Synth&egrave;se PGI(2) r&eacute;duite peut contribuer &agrave; d&eacute;p&ocirc;t plaquettaire local, accumulation de cellules musculaires lisses vasculaires, l&#39;ath&eacute;roscl&eacute;rose et finalement &eacute;chec des pontages veineux. Nous avons examin&eacute; si g&egrave;ne induite par le transfert de la surexpression de la COX-1 dans les veines greff&eacute;es (1) des augmentations PGI(2) et l&#39;AMP cyclique (AMPc) production, (2) conduit &agrave; une vasodilatation et la circulation sanguine locale am&eacute;lior&eacute;e en pr&eacute;sence de l&#39;hypercholest&eacute;rol&eacute;mie et (3) r&eacute;duit la formation n&eacute;o-intimale.

Transfert de g&egrave;nes de la COX-1 am&eacute;liore la taille de la lumi&egrave;re et le flux sanguin dans la carotide d&eacute;rivation greffes.

In autologous saphenous vein grafts, prostacyclin (PGI(1)), a vasoprotective molecule produced by normal endothelial cells, is down-regulated compared with ungrafted saphenous veins and normal carotid arteries. Reduced PGI(2) synthesis may contribute to local platelet deposition, vascular smooth muscle cell (VSMC) accumulation, atherosclerosis, and ultimately failure of venous bypass grafts. We have examined whether gene transfer-mediated overexpression of COX-1 in grafted veins (1) increases PGI(2) and cyclic AMP (cAMP) production, (2) leads to vasodilation and improved local blood flow in the presence of hypercholesterolemia, and (3) reduces neointima formation.

Gene transfer of COX-1 improves lumen size and blood flow in carotid bypass grafts.

Bone marrow cell therapy promotes angiogenesis, but the cellular fate of bone marrow cells (BMCs) in the absence of immunosuppressant interventions is unclear. We created a model of severe hind limb ischemia to address whether BMCs form new blood vessels or differentiate into other tissues.

Intra-arterial transplantation of adult bone marrow cells restores blood flow and regenerates skeletal muscle in ischemic limbs.

Th&eacute;rapie cellulaire de la moelle osseuse favorise l&#39;angiogen&egrave;se, mais le destin cellulaire des cellules de la moelle osseuse (BMC) en l&#39;absence d&#39;interventions de l&#39;immunosuppresseur n&#39;est pas clair. Nous avons cr&eacute;&eacute; un mod&egrave;le d&#39;isch&eacute;mie s&eacute;v&egrave;re membre post&eacute;rieur &agrave; l&#39;adresse si ca forme de nouveaux vaisseaux sanguins ou de se diff&eacute;rencier en d&#39;autres tissus.

Intra-art&eacute;rielle transplantation de cellules de la moelle osseuse adulte r&eacute;tablit la circulation sanguine et r&eacute;g&eacute;n&egrave;re le muscle squelettique des membres isch&eacute;mique.

Terapia cellulare del midollo osseo promuove l&#39;angiogenesi, ma non &egrave; chiaro il destino cellulare delle cellule del midollo osseo (BMC) in assenza di interventi di immunosoppressore. Abbiamo creato un modello di ischemia grave degli arti posteriori all&#39;indirizzo BMC formare nuovi vasi sanguigni o differenziarsi in altri tessuti.

Intra-arteriosa trapianto di cellule del midollo osseo adulto ripristina il flusso sanguigno e rigenera il muscolo scheletrico degli arti ischemici.

Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however, the optimal cell type and long-term efficacy are unknown. In this study, we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34âº/M-cadâº BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34âº/M-cadâº BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD.

CD34âº/M-cadherinâº bone marrow progenitor cells promote arteriogenesis in ischemic hindlimbs of ApoEâ»/â» mice.

La th&eacute;rapie cellulaire est prometteuse dans le traitement de la maladie art&eacute;rielle p&eacute;riph&eacute;rique (PAD) ; Cependant, le type de cellule optimale et l&#39;efficacit&eacute; &agrave; long terme sont inconnus. Dans cette &eacute;tude, nous avons identifi&eacute; une nouvelle sous-population de cellules prog&eacute;nitrices adultes positifs pour CD34 et M-cadh&eacute;rine (CD34&#x207A;/M-cad&#x207A; CR) chez la souris et la moelle osseuse humaine. Nous avons &eacute;galement examin&eacute; l&#39;efficacit&eacute; th&eacute;rapeutique de longue dur&eacute;e de souris CD34&#x207A;/M-cad&#x207A; CR en r&eacute;tablissant la circulation sanguine et la promotion de vascularisation dans un mod&egrave;le de souris ath&eacute;roscl&eacute;reuse de PAD.

CD34&#x207A;/M-cadherin&#x207A; la moelle osseuse des cellules prog&eacute;nitrices promouvoir arteriogenesis dans isch&eacute;mique membres post&eacute;rieurs des souris ApoE&#x207B; / &#x207B;.

Cell-based terapia Mostra la promessa nel trattamento della malattia arteriosa periferica (PAD); Tuttavia, il tipo di cella ottimale e l&#39;efficacia a lungo termine sono sconosciuti. In questo studio, abbiamo identificato una romanzo sottopopolazione di cellule progenitrici adulte positive per CD34 e M-caderina (CD34&#x207A;/M-cad&#x207A; BMC) nel topo e umano del midollo osseo. Abbiamo anche esaminato il duraturo effetto terapeutico del mouse CD34&#x207A;/M-cad&#x207A; BMC nel ripristino del flusso sanguigno e promuovere la vascolarizzazione in un modello murino aterosclerotica del PAD.

Cellule progenitrici del midollo osseo di CD34&#x207A;/M-cadherin&#x207A; promuovono arteriogenesis in ischemico posteriori dei topi ApoE&#x207B; / &#x207B;.

Prostacyclin (PGI2) is a potent vasodilator and important mediator of vascular homeostasis; however, its clinical use is limited because of its short (<2-min) half-life. Thus, we hypothesize that the use of engineered endothelial progenitor cells (EPCs) that constitutively secrete high levels of PGI2 may overcome this limitation of PGI2 therapy. A cDNA encoding COX-1-10aa-PGIS, which links human cyclooxygenase-1 (COX-1) to prostacyclin synthase (PGIS), was delivered via nucleofection into outgrowth EPCs derived from rat bone marrow mononuclear cells. PGI2-secreting strains (PGI2-EPCs) were established by continuous subculturing of transfected cells under G418 selection. Genomic PCR, RT-PCR, and Western blot analyses confirmed the overexpression of COX-1-10aa-PGIS in PGI2-EPCs. PGI2-EPCs secreted significantly higher levels of PGI2 in vitro than native EPCs (Pâ€‰<â€‰0.05) and showed higher intrinsic angiogenic capability; conditioned medium (CM) from PGI2-EPCs promoted better tube formation than CM from native EPCs (Pâ€‰<â€‰0.05). Cell- and paracrine-mediated in vitro angiogenesis was attenuated when COX-1-10aa-PGIS protein expression was knocked down. Whole-cell patch-clamp studies showed that 4-aminopyridine-sensitive K(+) current density was increased significantly in rat smooth muscle cells (rSMCs) cocultured under hypoxia with PGI2-EPCs (7.50â€‰Â±â€‰1.59 pA/pF; Pâ€‰<â€‰0.05) compared with rSMCs cocultured with native EPCs (3.99â€‰Â±â€‰1.26â€‰pA/pF). In conclusion, we successfully created EPC strains that overexpress an active novel enzyme resulting in consistent secretion of PGI2. PGI2-EPCs showed enhanced intrinsic proangiogenic properties and provided favorable paracrine-mediated cellular protections, including promoting in vitro angiogenesis of native EPCs and hyperpolarization of SMCs under hypoxia.

Engineered endothelial progenitor cells that overexpress prostacyclin protect vascular cells.

Prostacycline (PGI2) est un puissant vasodilatateur et un m&eacute;diateur important de l&#39;hom&eacute;ostasie vasculaire ; Cependant, son utilisation clinique est limit&eacute;e en raison de sa courte (&lt; 2-min) Half-Life. Ainsi, nous &eacute;mettons l&#39;hypoth&egrave;se que l&#39;utilisation de cellules prog&eacute;nitrices endoth&eacute;liales machin&eacute; (EPCs) qui s&eacute;cr&egrave;tent des niveaux &eacute;lev&eacute;s de PGI2 constitutivement peut surmonter cette limitation de PGI2 th&eacute;rapie. Un ADNc codant COX-1-10aa-IGP, qui relie les humains cyclo-oxyg&eacute;nase-1 (COX-1) prostacycline synthase (IGP), fourni via nucleofection en excroissance EPCs d&eacute;riv&eacute;es de cellules mononucl&eacute;aires de moelle osseuse de rats. Souches s&eacute;cr&eacute;trices de PGI2 (PGI2-EPCs) ont &eacute;t&eacute; &eacute;tablies par repiquage continu de cellules transfect&eacute;es sous s&eacute;lection G418. Analyses par transfert Western g&eacute;nomique PCR et RT-PCR a confirm&eacute; la surexpression de la COX-1-10aa-IGP en PGI2-EPCs. EPCs-PGI2 s&eacute;cr&eacute;t&eacute;es significativement plus &eacute;lev&eacute; de la PGI2 in vitro que natives EPCs (P 0,05 &lt;) et a montr&eacute; une plus grande capacit&eacute; angiog&eacute;nique intrins&egrave;que ; milieu conditionn&eacute; (CM) de PGI2-EPCs promu meilleure formation de tubes de CM du natifs EPCs (P 0,05 &lt;). M&eacute;diation cellulaire et paracrine-angiogen&egrave;se in vitro a &eacute;t&eacute; att&eacute;nu&eacute;e lorsque expression COX-1-10aa-IGP de la prot&eacute;ine a &eacute;t&eacute; renvers&eacute;e. Les &eacute;tudes de patch-clamp de la cellule enti&egrave;re ont montr&eacute; que cette densit&eacute; de courant de 4-aminopyridine sensible k a augment&eacute; consid&eacute;rablement dans les cellules de muscle lisse du rat (FFRS) cultiv&eacute;es sous une hypoxie avec PGI2-EPCs (7.50 &plusmn; 1,59 pA/pF ; P &lt; 0,05) par rapport aux FFRS co-cultiv&eacute;es avec natives EPCs (3,99 &plusmn; 1.26 pA/pF). En conclusion, nous avons cr&eacute;&eacute; avec succ&egrave;s des souches de CBE qui surexpriment une nouvelle enzyme active aboutissant &agrave; la s&eacute;cr&eacute;tion coh&eacute;rente de la PGI2. PGI2-EPCs a montr&eacute; des propri&eacute;t&eacute;s intrins&egrave;ques on am&eacute;lior&eacute;es et fourni favorables protections cellulaires m&eacute;di&eacute;e par paracrine, dont promotion angiogen&egrave;se in vitro de l&#39;EPCs natives et hyperpolarisation des CML en vertu de l&#39;hypoxie.

Cellules prog&eacute;nitrices endoth&eacute;liales machin&eacute; qui surexpriment prostacycline prot&egrave;gent les cellules vasculaires.

Prostaciclina (PGI2) &egrave; un potente vasodilatatore e importante Mediatore dell&#39;omeostasi vascolare; Tuttavia, il suo uso clinico &egrave; limitato a causa della sua breve (&lt; 2-min) Half-Life. Cos&igrave;, si ipotizza che l&#39;uso di cellule progenitrici endoteliali ingegnerizzati (EPC) che costitutivamente secernono alti livelli di PGI2 pu&ograve; superare questa limitazione della terapia PGI2. Un cDNA che codifica la COX-1-10aa-IGP, che collega umano cyclooxygenase-1 (COX-1) a prostaciclina sintasi (IGP), &egrave; stato trasportato via nucleofection in escrescenza EPCs derivato da cellule mononucleari di midollo osseo di ratto. PGI2-secernenti ceppi (PGI2-EPC) sono stati stabiliti da continuo subcoltura di celle transfected sotto selezione G418. Genomic PCR, RT-PCR e Western blot analisi ha confermato l&#39;iperespressione di COX-1-10aa-IGP in PGI2-EPC. PGI2-EPC secernuto significativamente pi&ugrave; elevati livelli di PGI2 in vitro rispetto al nativo EPCs (P &lt; 0.05) e ha mostrato intrinseca angiogenic pi&ugrave; elevate; condizionata media (CM) da PGI2-EPC promosso migliore formazione del tubo di CM dalla nativa EPCs (P &lt; 0,05). Cella - e paracrina-mediata angiogenesi in vitro &egrave; stato attenuato quando fu abbattuto l&#39;espressione della proteina COX-1-10aa-IGP. Cellule intere patch-clamp studi hanno mostrato che densit&agrave; di corrente K(+) 4-aminopiridina-sensibile &egrave; stato aumentato significativamente nelle cellule muscolari lisce di ratto (rSMCs) cocultured sotto l&#39;ipossia con PGI2-EPC (7,50 &plusmn; 1,59 pA/pF; P &lt; 0.05) rispetto ai rSMCs cocultured con nativo EPCs (pA/pF &plusmn; 3.99 1,26). In conclusione, abbiamo creato con successo ceppi EPC che iperesprimono un enzima attivo romanzo con conseguente coerenza secrezione di PGI2. PGI2-EPC ha mostrato enhanced proangiogenic intrinseche propriet&agrave; e condizione favorevole protezioni di cellulare paracrina-mediata, tra cui promuovere l&#39;angiogenesi in vitro di EPCs nativo e hyperpolarization di SMCs sotto l&#39;ipossia.

Le cellule progenitrici endoteliali ingegnerizzato che iperesprimono prostaciclina proteggono cellule vascolari.

Prostacyclin and its stable analogs play an important vascular protective role by promoting angiogenesis, but their role in arteriolar growth is unclear. Here, we examined the effect of prostacyclin stable analog carbaprostacyclin on arteriolar growth in mouse hindlimb ischemia. Using an osmotic-controlled release system to continuously deliver carbaprostacyclin or saline (control) to ischemic mouse hindlimbs for up to 14 days, we found that blood perfusion was significantly better at 7 and 14 days in carbaprostacyclin-treated mice than in saline-treated mice. Microscopic examination of the microvasculature showed more morphological signs of arteriolar formation in carbaprostacyclin- versus saline-treated legs. A double-blind, quantitative microcomputed tomography analysis indicated that carbaprostacyclin-treated legs had markedly increased vascular volume and small- to medium-sized vessel numbers that correspond to decreased vessel separation. A proteome profiler antibody array demonstrated that carbaprostacyclin-treated ischemic muscles secreted significantly higher amounts of acidic fibroblast growth factor and other chemokines. Conditioned media containing those secreted factors promoted smooth muscle cell growth and migration. Additionally, increased acidic fibroblast growth factor protein levels were detected in smooth muscle cells and skeletal myotubes at different time periods after carbaprostacyclin treatment. Furthermore, the selective peroxisome proliferation-activated receptor Î²/Î´ antagonist significantly suppressed carbaprostacyclin-induced acidic fibroblast growth factor protein production. Collectively, our data provide the first morphological and molecular evidence that local delivery of carbaprostacyclin promotes vascular growth in hindlimb ischemia, and that peroxisome proliferation-activated receptor Î²/Î´ signaling plays a critical role in inducing acidic fibroblast growth factor expression.

Targeted delivery of carbaprostacyclin to ischemic hindlimbs enhances adaptive remodeling of the microvascular network.

Prostacycline et ses analogues stables jouent un r&ocirc;le important de protection vasculaire en promouvant l&#39;angiogen&egrave;se, mais leur r&ocirc;le dans la croissance art&eacute;riolaire n&#39;est pas clair. Ici, nous avons examin&eacute; l&#39;effet de la prostacycline stable carbaprostacyclin analogiques sur croissance art&eacute;riolaire dans l&#39;isch&eacute;mie du membre post&eacute;rieur de la souris. Utilisez un syst&egrave;me osmotique-controlled release pour offrir continuellement carbaprostacyclin ou une solution saline (contr&ocirc;le) de souris isch&eacute;mique post&eacute;rieurs jusqu&#39;&agrave; 14 jours, nous avons constat&eacute; que la perfusion sanguine &eacute;tait significativement meilleure &agrave; 7 &agrave; 14 jours chez les souris trait&eacute;es carbaprostacyclin que chez les souris trait&eacute;es &agrave; une solution saline. L&#39;examen microscopique de la microcirculation a montr&eacute; plus de signes morphologiques de formation des art&eacute;rioles dans carbaprostacyclin et jambes impr&eacute;gn&eacute;es d&#39;une solution saline. Une analyse de tomographie quantitative en double-aveugle, microcomputed a indiqu&eacute; qu&#39;impr&eacute;gn&eacute;es de carbaprostacyclin jambes avaient nettement augment&eacute; de volume vasculaire et nombre de navires de petites &agrave; moyennes correspondant &agrave; une diminution s&eacute;paration de navire. Un tableau d&#39;anticorps de profileur proteome d&eacute;montr&eacute; que trait&eacute;s carbaprostacyclin isch&eacute;miques muscles s&eacute;cr&eacute;t&eacute;e des montants beaucoup plus &eacute;lev&eacute;s de facteur de croissance fibroblastique acides et autres chimiokines. Milieux conditionn&eacute;s contenant ces facteurs s&eacute;cr&eacute;t&eacute;s promu des migrations et la croissance des cellules musculaires lisses. En outre, taux de prot&eacute;ines de facteur de croissance fibroblastique acides accrue ont &eacute;t&eacute; d&eacute;tect&eacute;es dans les cellules musculaires lisses et des myotubes squelettiques &agrave; diff&eacute;rentes p&eacute;riodes de temps apr&egrave;s le traitement de carbaprostacyclin. En outre, l&#39;antagoniste &beta;/&delta; r&eacute;cepteur activ&eacute; par la prolif&eacute;ration des peroxysomes s&eacute;lective supprim&eacute;e significativement production de prot&eacute;ines pour le facteur de croissance fibroblastique acide induite par le carbaprostacyclin. Collectivement, nos r&eacute;sultats fournissent la premi&egrave;re preuve morphologique et mol&eacute;culaire que remise locale de carbaprostacyclin favorise la croissance vasculaire dans l&#39;isch&eacute;mie du membre post&eacute;rieur, et que ce r&eacute;cepteur activ&eacute; par la prolif&eacute;ration de peroxysomes &beta;/&delta; signalisation joue un r&ocirc;le crucial en induisant l&#39;expression du facteur de croissance fibroblastique acides.

CIBLEE de carbaprostacyclin aux membres post&eacute;rieurs isch&eacute;mique am&eacute;liore adaptative remodelage du r&eacute;seau microvasculaire.

Prostaciclina e suoi analoghi stabili giocano un importante ruolo protettivo vascolare promuovendo l&#39;angiogenesi, ma il loro ruolo nella crescita arteriolare &egrave; poco chiaro. Qui, abbiamo esaminato l&#39;effetto della prostaciclina stabile carbaprostacyclin analogico su arteriolare crescita nell&#39;ischemia arto posteriore del mouse. Utilizzando un sistema osmotico-controlled release per fornire continuamente carbaprostacyclin o soluzione salina (controllo) a posteriori ischemici del mouse fino a 14 giorni, abbiamo trovato che perfusione di sangue era significativamente migliore di 7 e 14 giorni nei topi trattati con carbaprostacyclin che nei topi trattati con soluzione fisiologica. L&#39;esame microscopico del microvasculature ha mostrato pi&ugrave; segni morfologici di formazione arteriolare in carbaprostacyclin-contro le gambe trattate con soluzione fisiologica. Un&#39;analisi di tomografia quantitativa in doppio cieco, microtomografia indicato che trattate con carbaprostacyclin gambe erano marcatamente aumentato volume vascolare e numeri vaso di piccole e medie dimensioni che corrispondono a diminuito separazione del vaso. Una matrice di anticorpo proteome profiler ha dimostrato che muscoli ischemici trattati carbaprostacyclin secernuto significativamente una maggiore quantit&agrave; di fattore di crescita dei fibroblasti acide e altre chemiochine. Media condizionata contenente quei fattori secrete promosso migrazione e crescita delle cellule muscolari lisce. Inoltre, livelli di proteina fattore di crescita del fibroblasto acida aumentata sono stati rilevati nelle cellule muscolari lisce e scheletriche myotubes a diversi periodi di tempo dopo il trattamento carbaprostacyclin. Inoltre, antagonista selettiva dei perossisomi attivato di proliferazione dei recettori &beta;/&delta; significativamente soppressa produzione di proteine del fattore di crescita dei fibroblasti acida indotta da carbaprostacyclin. Collettivamente, i nostri dati forniscono la prima prova morfologica e molecolare che consegna locale del carbaprostacyclin promuove la crescita vascolare nell&#39;ischemia arto posteriore, e quello dei perossisomi attivato di proliferazione recettori &beta;/&delta; segnalazione svolge un ruolo critico nell&#39;indurre l&#39;espressione di fattore di crescita dei fibroblasti acide.

Toya Terry

The Texas Heart Institute at St. Luke's Episcopal Hospital

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging