Afshin Ehsan

Isolation and Transplantation of Autologous Circulating Endothelial Cells Into Denuded Vessels and Prosthetic Grafts Implications for Cell-Based Vascular Therapy

Background—Blood-borne endothelial cells originating from adult bone marrow were reported previously. These cells have the properties of an endothelial progenitor cell (EPC) and can be mobilized by cytokines and recruited to sites of neovascularization, where they differentiate into mature endothelial cells. Current protocols for isolation of EPCs from peripheral blood rely on enrichment and selection of CD34+ mononuclear cells. Methods and Results—In this report, we describe a streamlined method for the isolation and expansion of EPCs from peripheral blood and evaluate their therapeutic potential for autologous cell-based therapy of injured blood vessels and prosthetic grafts. A subset of unfractionated mononuclear cells exhibited the potential to differentiate in vitro into endothelial cells under selective growth conditions. The cells were efficiently transduced ex vivo by a retroviral vector expressing the LacZ reporter gene and could be expanded to yield sufficient numbers for therapeutic applications. Transplantation of these cells into balloon-injured carotid arteries and into bioprosthetic grafts in rabbits led to rapid endothelialization of the denuded vessels and graft segments, resulting in significant reduction in neointima deposition. Conclusions—We conclude that transplantation of EPCs may play a crucial role in reestablishing endothelial integrity in injured vessels, thereby inhibiting neointimal hyperplasia. These findings may have implications for novel and practical cell-based therapies for vascular disease.

Ex-vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial

BACKGROUND Cell-cycle blockade by ex-vivo gene therapy of experimental vein grafts inhibits the neointimal hyperplasia and subsequent accelerated atherosclerosis that lead to human bypass-graft failure. In a prospective, randomised, controlled trial, we investigated the safety and biological efficacy of intraoperative gene therapy in patients receiving bypass vein grafts. METHODS We studied gene therapy that uses decoy oligodeoxynucleotide, which binds and inactivates the pivotal cell-cycle transcription factor E2F. 41 patients were randomly assigned untreated (16), E2F-decoy-treated (17), or scrambled-oligodeoxynucleotide-treated (eight) human infrainguinal vein grafts. Oligonucleotide was delivered to grafts intraoperatively by ex-vivo pressure-mediated transfection. The primary endpoints were safety and inhibition of target cell-cycle regulatory genes and of DNA synthesis in the grafts. Analysis was by intention to treat. FINDINGS Mean transfection efficiency was 89.0% (SD 1.9). Proliferating-cell nuclear antigen and c-myc mRNA concentrations and bromodeoxyuridine incorporation were decreased in the EF2-decoy group by medians of 73% [IQR 53-84], 70% [50-79], and 74% [56-83], respectively) but not in the scrambled-oligodeoxynucleotide group (p<0.0001). Groups did not differ for postoperative complication rates. At 12 months, fewer graft occlusions, revisions, or critical stenoses were seen in the E2F-decoy group than in the untreated group (hazard ratio 0.34 [95% CI 0.12-0.99]). INTERPRETATION Intraoperative transfection of human bypass vein grafts with E2F-decoy oligodeoxynucleotide is safe, feasible, and can achieve sequence-specific inhibition of cell-cycle gene expression and DNA replication. Application of this genetic-engineering strategy may lower failure rates of human primary bypass vein grafting.

Nodal metastasis and sites of recurrence after en bloc esophagectomy for adenocarcinoma

The operative specimens from 43 patients undergoing en bloc esophagectomy for adenocarcinoma of the lower esophagus or cardia were analyzed. Depth of invasion of the tumor and extent and location of lymph node metastases were determined. Postoperative recurrence was identified from positive findings on successive 3-month computed tomographic scans. Positive nodes occurred in 33% (2/6) of intramucosal tumors, 67% (6/9) of intramural tumors, and 89% (25/28) of transmural tumors (p < 0.01). Commonly involved nodes were those in the lesser curve of the stomach (42%), parahiatal nodes (35%), paraesophageal nodes (28%), and celiac nodes (21%). Excluding perioperative deaths, follow-up was complete for 38 patients. Twenty patients had recurrence. Fifteen patients (40%, 15/38) had nodal recurrence: cervical, 7.9% (3/38); superior mediastinal, 21% (8/38); and abdominal, 24% (9/38) (retropancreatic in 7 and retrocrural in 2). Of 5 patients with nodal recurrence alone, 3 (60%) had recurrence at sites outside the margins of resection. Patients with four metastatic nodes or less had a survival advantage over those with more than four (p < 0.05). There was no difference in survival according to location of nodal metastases. Two (22.2%) of 9 patients with celiac node metastases survived longer than 4 years. Adenocarcinoma of the lower esophagus and cardia spreads widely to mediastinal and abdominal nodes, and death can occur from nodal disease. Rates of lymph node metastases increase with the depth of the primary tumor. Patients with lymphatic metastases can be cured particularly if there are fewer than four nodes involved. Curative surgical therapy necessitates wide lymph node resection to ensure removal of all metastatic nodes.

Focused ultrasound (HIFU) induces localized enhancement of reporter gene expression in rabbit carotid artery

The development of accurate, safe, and efficient gene delivery remains a major challenge towards the realization of gene therapeutic prevention and treatment of cardiovascular diseases. In this study, we investigated the ability of high-intensity focused ultrasound (HIFU), a form of mechanical wave transmission, to act as a noninvasive tool for the enhancement of in vivo gene transfer into rabbit carotid arteries. Segments of the common carotid arteries of New Zealand white rabbits were isolated and infused with plasmid DNA encoding the reporter β-galactosidase either with or without the addition of ultrasound contrast agent consisting of small (∼2–5 μm) gas-filled human albumin microspheres to augment cavitation. Infused arteries were exposed to pulsed ultrasound for 1 min (frequency 0.85 MHz, burst length 50 ms, repetition frequency 1 Hz, duration 60 s, peak pressure amplitude of 15 MPa). At 6.3 MPa, HIFU enhanced gene expression eight-fold, and 17.5-fold in the presence of contrast. We found increasing amounts of β-galactosidase expression in the carotid vessel with increasing pressure amplitude. This dose–response relation was present with and without contrast. Without contrast, no vessel damage was detected up to 15 MPa, while the addition of contrast induced side effects above a threshold of 6.3 MPa peak pressure. The entire procedure was feasible and safe for the animals, and the results suggest that HIFU has the potential to assist in the noninvasive spatial regulation of gene transfer into the vascular system.

Endothelial Healing in Vein Grafts Proliferative Burst Unimpaired by Genetic Therapy of Neointimal Disease

Background—Although inhibition of neointimal hyperplasia by cell cycle gene blockade therapy results in improved endothelial cell function in experimental vein grafts, little is known either about endothelial healing immediately after vein grafting or about the effect of this therapy on the healing process. Methods and Results—Scanning electron microscopy demonstrated an immediate decrease in vein graft endothelial cell density associated with vein graft wall stretch, followed by a return to baseline by postoperative day 3. En face detection of bromodeoxyuridine incorporation confirmed a rapid endothelial proliferation by 48 hours. Despite inhibition of underlying vascular smooth muscle cell proliferation, E2F decoy oligonucleotide did not inhibit either endothelial bromodeoxyuridine incorporation or the return to baseline cell density. This differential response to E2F decoy was also observed in human umbilical vein endothelial cell culture, which resisted the E2F decoy inhibition of cell growth that was observed in human umbilical artery smooth muscle cells, despite evidence for nuclear localized delivery of the oligonucleotide into both cell types. Furthermore, the reduction of E2F binding activity seen in a nuclear gel shift assay of cultured smooth muscle cells was not observed in endothelial cells. Conclusions—These results suggest a burst of graft endothelial cell proliferation that allows a rapid restoration of cell density in the monolayer. Additionally, there is a selective effect of E2F decoy gene therapy on target smooth muscle cells with sparing of this endothelial healing.

Placental growth factor mediates aldosterone-dependent vascular injury in mice

In clinical trials, aldosterone antagonists reduce cardiovascular ischemia and mortality by unknown mechanisms. Aldosterone is a steroid hormone that signals through renal mineralocorticoid receptors (MRs) to regulate blood pressure. MRs are expressed and regulate gene transcription in human vascular cells, suggesting that aldosterone might have direct vascular effects. Using gene expression profiling, we identify the pro-proliferative VEGF family member placental growth factor (PGF) as an aldosterone-regulated vascular MR target gene in mice and humans. Aldosterone-activated vascular MR stimulated Pgf gene transcription and increased PGF protein expression and secretion in the mouse vasculature. In mouse vessels with endothelial damage and human vessels from patients with atherosclerosis, aldosterone enhanced expression of PGF and its receptor, FMS-like tyrosine kinase 1 (Flt1). In atherosclerotic human vessels, MR antagonists inhibited PGF expression. In vivo, aldosterone infusion augmented vascular remodeling in mouse carotids following wire injury, an effect that was lost in Pgf-/- mice. In summary, we have identified PGF as what we believe to be a novel downstream target of vascular MR that mediates aldosterone augmentation of vascular injury. These findings suggest a non-renal mechanism for the vascular protective effects of aldosterone antagonists in humans and support targeting the vascular aldosterone/MR/PGF/Flt1 pathway as a therapeutic strategy for ischemic cardiovascular disease.

Aldosterone Regulates Vascular Gene Transcription via Oxidative Stress–Dependent and –Independent Pathways

Objective—Aldosterone (Aldo) antagonism prevents cardiovascular mortality by unclear mechanisms. Aldo binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor, which is expressed in human vascular cells. Here we define the early Aldo-regulated vascular transcriptome and investigate the mechanisms of gene regulation by Aldo in the vasculature that may contribute to vascular disease. Methods and Results—Gene expression profiling of Aldo-treated mouse aortas identified 72 genes regulated by Aldo. These genes are overrepresented in Gene Ontology categories involved in vascular function and disease. Quantitative reverse transcription–polymerase chain reaction was used to confirm and further explore mechanisms of vascular gene regulation by Aldo. Aldo-regulated vascular gene expression was inhibited by actinomycin D and MR antagonists supporting a transcriptional MR-dependent mechanism. Aldo regulation of a subset of genes was enhanced in the setting of vascular endothelial denudation and blocked by the free radical scavenger Tempol, supporting synergy between Aldo and vascular injury that is oxidative stress dependent. In the aortic arch, a region predisposed to atherosclerosis, the injury-enhanced genes also demonstrated enhanced expression compared with the descending aorta, both at baseline and after Aldo exposure. Furthermore, the clinically beneficial MR antagonist spironolactone inhibited expression of the identified genes in aortic tissue from humans with atherosclerosis. Conclusion—This study defines the Aldo-regulated vascular transcriptome and characterizes a subset of proatherogenic genes with enhanced Aldo-stimulated, oxidative stress–dependent expression in the setting of vascular injury and in areas predisposed to atherosclerosis. Inhibition of MR regulation of these genes may play a role in the protective effects of Aldo antagonists in patients with vascular disease, and these pathways may provide novel drug targets to prevent atherosclerosis in humans.

Posttranscriptional regulation of angiotensin II type 1 receptor expression by glyceraldehyde 3-phosphate dehydrogenase

Regulation of angiotensin II type 1 receptor (AT1R) has a pathophysiological role in hypertension, atherosclerosis and heart failure. We started from an observation that the 3′-untranslated region (3′-UTR) of AT1R mRNA suppressed AT1R translation. Using affinity purification for the separation of 3′-UTR-binding proteins and mass spectrometry for their identification, we describe glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an AT1R 3′-UTR-binding protein. RNA electrophoretic mobility shift analysis with purified GAPDH further demonstrated a direct interaction with the 3′-UTR while GAPDH immunoprecipitation confirmed this interaction with endogenous AT1R mRNA. GAPDH-binding site was mapped to 1–100 of 3′-UTR. GAPDH-bound target mRNAs were identified by expression array hybridization. Analysis of secondary structures shared among GAPDH targets led to the identification of a RNA motif rich in adenines and uracils. Silencing of GAPDH increased the expression of both endogenous and transfected AT1R. Similarly, a decrease in GAPDH expression by H2O2 led to an increased level of AT1R expression. Consistent with GAPDH having a central role in H2O2-mediated AT1R regulation, both the deletion of GAPDH-binding site and GAPDH overexpression attenuated the effect of H2O2 on AT1R mRNA. Taken together, GAPDH is a translational suppressor of AT1R and mediates the effect of H2O2 on AT1R mRNA.

Inactivation of Endothelial Small/Intermediate Conductance of Calcium‐Activated Potassium Channels Contributes to Coronary Arteriolar Dysfunction in Diabetic Patients

Background Diabetes is associated with coronary arteriolar endothelial dysfunction. We investigated the role of the small/intermediate (SKCa/IKCa) conductance of calcium-activated potassium channels in diabetes-related endothelial dysfunction. Methods and Results Coronary arterioles (80 to 150 μm in diameter) were dissected from discarded right atrial tissues of diabetic (glycosylated hemoglobin = 9.6±0.25) and nondiabetic patients (glycosylated hemoglobin 5.4±0.12) during coronary artery bypass graft surgery (n=8/group). In-vitro relaxation response of precontracted arterioles was examined in the presence of the selective SKCa/IKCa activator NS309 and other vasodilatory agents. The channel density and membrane potential of diabetic and nondiabetic endothelial cells was measured by using the whole cell patch-clamp technique. The protein expression and distribution of the SKCa/IKCa in the human myocardium and coronary arterioles was examined by Western blotting and immunohistochemistry. Our results indicate that diabetes significantly reduced the coronary arteriolar response to the SKCa/IKCa activator NS309 compared to the respective responses of nondiabetic vessels (P<0.05 versus nondiabetes). The relaxation response of diabetic arterioles to NS309 was prevented by denudation of endothelium (P=0.001 versus endothelium-intact). Diabetes significantly decreased endothelial SKCa/IKCa currents and hyperpolarization induced by the SKCa/IKCa activator NS309 as compared with that of nondiabetics. There were no significant differences in the expression and distribution of SKCa/IKCa proteins in the coronary microvessels. Conclusions Diabetes is associated with inactivation of endothelial SKCa/IKCa channels, which may contribute to endothelial dysfunction in diabetic patients.

Communication gaps associated with donor-derived infections

The detection and management of potential donor‐derived infections is challenging, in part due to the complexity of communications between diverse labs, organ procurement organizations (OPOs), and recipient transplant centers. We sought to determine if communication delays or errors occur in the reporting and management of donor‐derived infections and if these are associated with preventable adverse events in recipients. All reported potential donor‐derived transmission events reviewed by the Organ Procurement and Transplantation Network Ad Hoc Disease Transmission Advisory Committee from January 2008 to June 2010 were evaluated for communication gaps between the donor center, OPO and transplant centers. The impact on recipient outcomes was then determined. Fifty‐six infection events (IEs; involving 168 recipients) were evaluated. Eighteen IEs (48 recipients) were associated with communication gaps, of which 12 resulted in adverse effects in 69% of recipients (20/29), including six deaths. When IEs and test results were reported without delay, appropriate interventions were taken, subsequently minimizing or averting recipient infection (23 IEs, 72 recipients). Communication gaps in reported IEs are frequent, occur at multiple levels in the communication process, and contribute to adverse outcomes among affected transplant recipients. Conversely, effective communication minimized or averted infection in transplant recipients.