Early Injury Landscape in Vein Harvest by Single-Cell and Spatial Transcriptomics.

BACKGROUND: Vein graft failure following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. Although previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on vein graft failure. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. METHODS: Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing and spatial transcriptomics analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-carotid vein bypass implantation in a canine model (n=4). RESULTS: Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P<0.05) involved in the activation of endothelial cells (ECs), fibroblasts, and vascular smooth muscle cells, namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and ECM (extracellular matrix) remodeling throughout the vein wall. Subsequent single-nuclei RNA-sequencing analysis supported these findings and further unveiled distinct EC and fibroblast subpopulations with significant upregulation (P<0.05) of markers related to endothelial injury response and cellular activation of ECs, fibroblasts, and vascular smooth muscle cells. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN, FBN1, and VEGFC, in addition to novel genes of interest, such as GLIS3 and EPHA3. These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the spatial transcriptomics and single-nuclei RNA-sequencing data sets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and fibroblasts were notably enriched in the intima and media of distended veins. Finally, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal-transitioning ECs, protomyofibroblasts, and vascular smooth muscle cells in upregulating signaling pathways associated with cellular proliferation (MDK [midkine], PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor]), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. CONCLUSIONS: Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.

Integrated single-nuclei and spatial transcriptomic analysis reveals propagation of early acute vein harvest and distension injury signaling pathways following arterial implantation.

Background: Vein graft failure (VGF) following cardiovascular bypass surgery results in significant patient morbidity and cost to the healthcare system. Vein graft injury can occur during autogenous vein harvest and preparation, as well as after implantation into the arterial system, leading to the development of intimal hyperplasia, vein graft stenosis, and, ultimately, bypass graft failure. While previous studies have identified maladaptive pathways that occur shortly after implantation, the specific signaling pathways that occur during vein graft preparation are not well defined and may result in a cumulative impact on VGF. We, therefore, aimed to elucidate the response of the vein conduit wall during harvest and following implantation, probing the key maladaptive pathways driving graft failure with the overarching goal of identifying therapeutic targets for biologic intervention to minimize these natural responses to surgical vein graft injury. Methods: Employing a novel approach to investigating vascular pathologies, we harnessed both single-nuclei RNA-sequencing (snRNA-seq) and spatial transcriptomics (ST) analyses to profile the genomic effects of vein grafts after harvest and distension, then compared these findings to vein grafts obtained 24 hours after carotid-cartoid vein bypass implantation in a canine model (n=4). Results: Spatial transcriptomic analysis of canine cephalic vein after initial conduit harvest and distention revealed significant enrichment of pathways (P < 0.05) involved in the activation of endothelial cells (ECs), fibroblasts (FBs), and vascular smooth muscle cells (VSMCs), namely pathways responsible for cellular proliferation and migration and platelet activation across the intimal and medial layers, cytokine signaling within the adventitial layer, and extracellular matrix (ECM) remodeling throughout the vein wall. Subsequent snRNA-seq analysis supported these findings and further unveiled distinct EC and FB subpopulations with significant upregulation (P < 0.00001) of markers related to endothelial injury response and cellular activation of ECs, FBs, and VSMCs. Similarly, in vein grafts obtained 24 hours after arterial bypass, there was an increase in myeloid cell, protomyofibroblast, injury-response EC, and mesenchymal-transitioning EC subpopulations with a concomitant decrease in homeostatic ECs and fibroblasts. Among these markers were genes previously implicated in vein graft injury, including VCAN (versican), FBN1 (fibrillin-1), and VEGFC (vascular endothelial growth factor C), in addition to novel genes of interest such as GLIS3 (GLIS family zinc finger 3) and EPHA3 (ephrin-A3). These genes were further noted to be driving the expression of genes implicated in vascular remodeling and graft failure, such as IL-6, TGFBR1, SMAD4, and ADAMTS9. By integrating the ST and snRNA-seq datasets, we highlighted the spatial architecture of the vein graft following distension, wherein activated and mesenchymal-transitioning ECs, myeloid cells, and FBs were notably enriched in the intima and media of distended veins. Lastly, intercellular communication network analysis unveiled the critical roles of activated ECs, mesenchymal transitioning ECs, protomyofibroblasts, and VSMCs in upregulating signaling pathways associated with cellular proliferation (MDK, PDGF, VEGF), transdifferentiation (Notch), migration (ephrin, semaphorin), ECM remodeling (collagen, laminin, fibronectin), and inflammation (thrombospondin), following distension. Conclusions: Vein conduit harvest and distension elicit a prompt genomic response facilitated by distinct cellular subpopulations heterogeneously distributed throughout the vein wall. This response was found to be further exacerbated following vein graft implantation, resulting in a cascade of maladaptive gene regulatory networks. Together, these results suggest that distension initiates the upregulation of pathological pathways that may ultimately contribute to bypass graft failure and presents potential early targets warranting investigation for targeted therapies. This work highlights the first applications of single-nuclei and spatial transcriptomic analyses to investigate venous pathologies, underscoring the utility of these methodologies and providing a foundation for future investigations.

Lifelong limb preservation: A patient-centered description of lower extremity arterial reconstruction outcomes.

BACKGROUND: Life expectancy is short for patients with critical limb ischemia (CLI), many of whom may fear amputation more than death. In light of the reduced life expectancy of these patients, the traditional 5-year freedom from amputation (FFA) statistic may not accurately address their concern. We developed a more relevant patient-centered calculation of major amputation risk during a patient's remaining lifetime to better answer the question, Will I ever lose my leg? METHODS: We identified all limbs undergoing first-time intervention for CLI in a large institutional database from 2005 to 2013. We calculated the traditional metrics of amputation-free survival (AFS, for which failure is death or amputation) and FFA (for which failure is amputation but deaths are censored and removed from further analysis). In addition, we propose a new term, lifelong limb preservation (LLP). LLP defines amputation as failure, but deaths are not censored and therefore reflect that LLP has been achieved. All deaths before 30 days were considered a failure in all three metrics, reflecting the risk of surgery. RESULTS: There were 1006 limbs identified as having first-time intervention for CLI (22% rest pain, 45% ulcer, 27% gangrene; 46% treated by angioplasty with or without stenting, 54% bypass). Using life-table analysis, 7-year AFS was 14% (561 events), FFA was 78% (123 events), and LLP was 86% (123 events). LLP was similar between patients undergoing angioplasty with or without stenting and bypass (7-year rates, 86% and 85%, respectively). For patients undergoing intervention for rest pain, 7-year rates were 14% for AFS, 84% for FFA, and 92% for LLP. For those undergoing treatment for ulcer, 7-year rates were 14% for AFS, 77% for FFA, and 86% for LLP. Finally, in those with gangrene, rates were 10% for AFS, 67% for FFA, and 79% for LLP. Using LLP, patients presenting with an ulcer can be told that although we cannot guarantee how long they will live, with revascularization there is approximately an 86% chance they will not lose the leg. CONCLUSIONS: These results show that the durability of our limb preservation efforts often exceeds the life expectancy of our patients. Using LLP as an outcomes assessment provides a more accurate and patient-centered answer to the question, If I have this procedure, will I ever lose my leg?

Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.

In an effort to inhibit the response to vascular injury that leads to intimal hyperplasia, this study investigated the in vivo efficacy of intraluminal delivery of thrombospondin-2 (TSP-2) small interfering RNA (siRNA). Common carotid artery (CCA) balloon angioplasty injury was performed in rats. Immediately after denudation, CCA was transfected intraluminally (15 min) with one of the following: polyethylenimine (PEI)+TSP-2 siRNA, saline, PEI only, or PEI+control siRNA. CCA was analyzed at 24 h or 21 d by using quantitative real-time PCR and immunohistochemistry. TSP-2 gene and protein expression were significantly up-regulated after endothelial denudation at 24 h and 21 d compared with contralateral untreated, nondenuded CCA. Treatment with PEI+TSP-2 siRNA significantly suppressed TSP-2 gene expression (3.1-fold) at 24 h and TSP-2 protein expression, cell proliferation, and collagen deposition up to 21 d. These changes could be attributed to changes in TGF-ß and matrix metalloproteinase-9, the downstream effectors of TSP-2. TSP-2 knockdown induced anti-inflammatory M2 macrophage polarization at 21 d; however, it did not significantly affect intima/media ratios. In summary, these data demonstrate effective siRNA transfection of the injured arterial wall and provide a clinically effective and translationally applicable therapeutic strategy that involves nonviral siRNA delivery to ameliorate the response to vascular injury.-Bodewes, T. C. F., Johnson, J. M., Auster, M., Huynh, C., Muralidharan, S., Contreras, M., LoGerfo, F. W., Pradhan-Nabzdyk, L. Intraluminal delivery of thrombospondin-2 small interfering RNA inhibits the vascular response to injury in a rat carotid balloon angioplasty model.

Substance P promotes wound healing in diabetes by modulating inflammation and macrophage phenotype.

Diabetic foot ulceration is a major complication of diabetes. Substance P (SP) is involved in wound healing, but its effect in diabetic skin wounds is unclear. We examined the effect of exogenous SP delivery on diabetic mouse and rabbit wounds. We also studied the impact of deficiency in SP or its receptor, neurokinin-1 receptor, on wound healing in mouse models. SP treatment improved wound healing in mice and rabbits, whereas the absence of SP or its receptor impaired wound progression in mice. Moreover, SP bioavailability in diabetic skin was reduced as SP gene expression was decreased, whereas the gene expression and protein levels of the enzyme that degrades SP, neutral endopeptidase, were increased. Diabetes and SP deficiency were associated with absence of an acute inflammatory response important for wound healing progression and instead revealed a persistent inflammation throughout the healing process. SP treatment induced an acute inflammatory response, which enabled the progression to the proliferative phase and modulated macrophage activation toward the M2 phenotype that promotes wound healing. In conclusion, SP treatment reverses the chronic proinflammatory state in diabetic skin and promotes healing of diabetic wounds.

Current siRNA targets in the prevention and treatment of intimal hyperplasia.

Intimal hyperplasia (IH) is the leading cause of late vein and prosthetic bypass graft failure. Injury at the time of graft implantation leading to the activation of endothelial cells and dedifferentiation of vascular smooth muscle cells to a synthetic phenotype are known causes of IH. Prior attempts to develop therapy to mitigate these cellular changes to prevent IH and graft failure have failed. Small interfering RNA (siRNA) mediated targeted gene silencing is a promising tool to prevent IH. Several studies have been performed in this direction to target genes that are involved in IH. In this review we discuss siRNA targets that are being investigated for prevention and treatment of IH.

Gene silencing in human aortic smooth muscle cells induced by PEI-siRNA complexes released from dip-coated electrospun poly(ethylene terephthalate) grafts.

An excessive tissue response to prosthetic arterial graft material leads to intimal hyperplasia (IH), the leading cause of late graft failure. Seroma and abnormal capsule formation may also occur after prosthetic material implantation. The matricellular protein Thrombospondin-2 (TSP-2) has shown to be upregulated in response to biomaterial implantation. This study evaluates the uptake and release of small interfering RNA (siRNA) from unmodified and surface functionalized electrospun PET graft materials. ePET graft materials were synthesized using electrospinning technology. Subsets of the ePET materials were then chemically modified to create surface functional groups. Unmodified and surface-modified ePET grafts were dip-coated in siRNAs alone or siRNAs complexed with transfection reagents polyethyleneimine (PEI) or Lipofectamine RNAiMax. Further, control and TSP-2 siRNA-PEI complex treated ePET samples were placed onto a confluent layer of human aortic smooth muscle cells (AoSMCs). Complexation of all siRNAs with PEI led to a significant increase in adsorption to unmodified ePET. TSP-2 siRNA-PEI released from unmodified-ePET silenced TSP-2 in AoSMC. Regardless of the siRNA-PEI complex evaluated, AoSMC migrated into the ePET. siRNA-PEI complexes delivered to AoSMC from dip-coated ePET can result in gene knockdown. This methodology for siRNA delivery may improve the tissue response to vascular and other prosthetics.

Expression of neuropeptides and cytokines in a rabbit model of diabetic neuroischemic wound healing.

OBJECTIVE: The present study is designed to understand the contribution of peripheral vascular disease and peripheral neuropathy to the wound-healing impairment associated with diabetes. Using a rabbit model of diabetic neuroischemic wound healing, we investigated rate of healing, leukocyte infiltration, and expression of cytokines, interleukin-8 and interleukin-6, and neuropeptides, substance P, and neuropeptide Y. METHODS: Diabetes was induced in New Zealand White rabbits by administering alloxan while control rabbits received saline. Ten days later, animals in both groups underwent surgery. One ear served as a sham, and the other was made ischemic (ligation of central+rostral arteries) or neuroischemic (ischemia+ resection of central+rostral nerves). Four 6-mm punch biopsy wounds were created in both ears and wound healing was followed for 10 days using computerized planimetry. RESULTS: Nondiabetic sham and ischemic wounds healed significantly more rapidly than diabetic sham and ischemic wounds. Healing was slowest in neuroischemic wounds, irrespective of diabetic status. A high M1/M2 macrophage ratio and a high proinflammatory cytokine expression, both indicators of chronic proinflammatory state, and low neuropeptide expression were seen in preinjury diabetic skin. Postinjury, in diabetic wounds, the M1/M2 ratio remained high, the reactive increase in cytokine expression was low, and neuropeptide expression was further decreased in neuroischemic wounds. CONCLUSIONS: This rabbit model illustrates how a combination of a high M1/M2 ratio, a failure to mount postinjury cytokine response as well as a diminished neuropeptide expression, contribute to wound-healing impairment in diabetes. The addition of neuropathy to ischemia leads to equivalently severe impaired wound-healing irrespective of diabetes status, suggesting that in the presence of ischemia, loss of neuropeptide function contributes to the impaired healing associated with diabetes.

Temporal network based analysis of cell specific vein graft transcriptome defines key pathways and hub genes in implantation injury.

Vein graft failure occurs between 1 and 6 months after implantation due to obstructive intimal hyperplasia, related in part to implantation injury. The cell-specific and temporal response of the transcriptome to vein graft implantation injury was determined by transcriptional profiling of laser capture microdissected endothelial cells (EC) and medial smooth muscle cells (SMC) from canine vein grafts, 2 hours (H) to 30 days (D) following surgery. Our results demonstrate a robust genomic response beginning at 2 H, peaking at 12-24 H, declining by 7 D, and resolving by 30 D. Gene ontology and pathway analyses of differentially expressed genes indicated that implantation injury affects inflammatory and immune responses, apoptosis, mitosis, and extracellular matrix reorganization in both cell types. Through backpropagation an integrated network was built, starting with genes differentially expressed at 30 D, followed by adding upstream interactive genes from each prior time-point. This identified significant enrichment of IL-6, IL-8, NF-κB, dendritic cell maturation, glucocorticoid receptor, and Triggering Receptor Expressed on Myeloid Cells (TREM-1) signaling, as well as PPARα activation pathways in graft EC and SMC. Interactive network-based analyses identified IL-6, IL-8, IL-1α, and Insulin Receptor (INSR) as focus hub genes within these pathways. Real-time PCR was used for the validation of two of these genes: IL-6 and IL-8, in addition to Collagen 11A1 (COL11A1), a cornerstone of the backpropagation. In conclusion, these results establish causality relationships clarifying the pathogenesis of vein graft implantation injury, and identifying novel targets for its prevention.

Expression of Neuropeptide Y, Substance P, and their receptors in the right atrium of diabetic patients.

OBJECTIVE: To investigate the expression of neuropeptides and their receptors that play a role in cardiac homeostasis in the right atrium of nondiabetic and diabetic patients undergoing coronary artery bypass graft surgery. BACKGROUND: The cardioactive neuropeptides and their receptors investigated in this study were Neuropeptide Y (NPY), and its receptors, NPY Receptor1 (NPY1R), NPY Receptor2 (NPY2R), NPY Receptor5 (NPY5R) and Substance P (SP) and its receptor, Neurokinin1R (NK1R). METHODS: The gene and protein expression of NPY, NPY1R, NPY2R, NPY5R, SP and NK1R from the atrial tissue of 10 nondiabetic and diabetic patients undergoing coronary artery bypass grafting (CABG) was assessed by Q-RTPCR, immunohistochemistry, Western blot, and ELISA. RESULTS: Gene expression of NPY2R, NPY5R, preproTachykinin A (SP gene), and NK1R and their respective protein expression were significantly reduced whereas that of NPY and NPY1R were unchanged in the right atrium of diabetic patients compared to nondiabetic patients. CONCLUSIONS: These results demonstrate that the expression of neuropeptides and their receptors in the diabetic heart is significantly impaired, and may be the link between neuropathy and cardiac complications. Further studies are warranted to delineate pathophysiologic mechanisms associated with dysregulation of the cardiac neuropeptide system and the relationship to cardiac complications in diabetes.

Effect of hyperglycemia and neuropeptides on interleukin-8 expression and angiogenesis in dermal microvascular endothelial cells.

BACKGROUND: Impaired wound healing is a major complication associated with diabetes, involving a dysregulation and impairments in the inflammatory and angiogenic phases of wound healing. Here, we examine the effects of the neuropeptides substance P (SP) and neuropeptide Y (NPY) on dermal microvascular endothelial cell (DMVEC) angiogenesis and interleukin-8 (IL-8) expression, a known effector of the neuropeptide pathways in normal and hyperglycemic conditions in vitro. METHODS: DMVECs are treated with one of four glucose concentrations: 1) 5 mM glucose; 2) 10 mM glucose; 3) 30 mM glucose; or 4) 30 mM mannitol and cotreated with 100 nM NPY, 100 nM SP, or 10 ng/mL IL-8. Angiogenesis is assessed with proliferation and tube formation assays. IL-8 mRNA and protein expression are evaluated at days 1 and 7. RESULTS: As compared with noromoglycemia (5 mM glucose), hyperglycemia (30 mM glucose) decreases DMVEC proliferation and tube formation by 39% and 42%, respectively. SP cotreatment restores DMVEC proliferation (211%) and tube formation (152%), and decreases IL-8 expression (34%) in DMVECs exposed to hyperglycemic conditions. These effects are not observed with NPY. However, IL-8 treatment by itself does not affect proliferation or tube formation, suggesting that the effect of SP on DMVEC angiogenesis is unlikely through changes in IL-8 expression. CONCLUSION: Hyperglycemic conditions impair DMVEC proliferation and tube formation. SP mitigates the effect of hyperglycemia on DMVECs by increasing DMVEC proliferation and tube formation. These findings are not likely to be related to a dysregulation of IL-8 due to the lack of effects of hyperglycemia on IL-8 expression and the lack of effect of IL-8 on DMVEC proliferation and tube formation. The effect of SP on DMVECs makes SP a promising potential target for therapy in impaired wound healing in diabetes, but the exact mechanism remains unknown.

Gene expression of pro-inflammatory cytokines and neuropeptides in diabetic wound healing.

The interaction between neuropeptides and cytokines and its role in cutaneous wound healing is becoming evident. The goal of the present study is to investigate the impact of diabetes on peripheral cytokine and neuropeptide expression and its role in diabetic wound healing. To achieve this goal, the effect of diabetes on wound healing, along with the role of inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8) secreted in the wound microenvironment, and neuropeptides such as substance P (SP) and neuropeptide Y (NPY), secreted from peripheral nerves is monitored in non-diabetic and diabetic rabbits. Rabbits in the diabetic group received alloxan monohydrate (100mg/kg i.v.). Ten days after diabetic induction, four full thickness circular wounds were created in both ears using a 6mm punch biopsy. Wound healing was monitored over 10 d and gene expression of cytokines and neuropeptides was assessed in the wounds. Compared with the non-diabetic rabbits, wounds of diabetic rabbits heal significantly slower. Diabetic rabbits show significantly increased baseline gene expression of IL-6 and IL-8, their receptors, CXCR1, CXCR2, GP-130, and a decrease of prepro tachykinin-A (PP-TA), the precursor of SP, whereas the expression of prepro-NPY (PP-NPY), the precursor of NPY is not different. Similarly, baseline protein expression of CXCR1 is higher in diabetic rabbit skin. Post-injury, the increase over baseline gene expression of IL-6, IL-8, CXCR1, CXCR2, and GP-130 is significantly less in diabetic wounds compared with non-diabetic wounds. Although there is no difference in PP-TA gene expression between non-diabetic and diabetic rabbits post-injury, the gene expression of PP-NPY is reduced in diabetic rabbits. In conclusion, diabetes causes dysregulation in the neuropeptide expression in the skin along with a suppressed focused inflammatory response to injury. This suggests that the chronic inflammation in the skin of diabetic rabbits inhibits the acute inflammation much needed for wound healing.

Endothelial cells are susceptible to rapid siRNA transfection and gene silencing ex vivo.

BACKGROUND: Endothelial gene silencing via small interfering RNA (siRNA) transfection represents a promising strategy for the control of vascular disease. Here, we demonstrate endothelial gene silencing in human saphenous vein using three rapid siRNA transfection techniques amenable for use in the operating room. METHODS: Control siRNA, Cy5 siRNA, or siRNA targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or endothelial specific nitric oxide synthase (eNOS) were applied to surplus human saphenous vein for 10 minutes by (i) soaking, (ii) applying 300 mm Hg hyperbaric pressure, or (iii) 120 mm Hg luminal distending pressure. Transfected vein segments were maintained in organ culture. siRNA delivery and gene silencing were assessed by tissue layer using confocal microscopy and immunohistochemistry. RESULTS: Distending pressure transfection yielded the highest levels of endothelial siRNA delivery (22% pixels fluorescing) and gene silencing (60% GAPDH knockdown, 55% eNOS knockdown) as compared with hyperbaric (12% pixels fluorescing, 36% GAPDH knockdown, 30% eNOS knockdown) or non-pressurized transfections (10% pixels fluorescing, 30% GAPDH knockdown, 25% eNOS knockdown). Cumulative endothelial siRNA delivery (16% pixels fluorescing) and gene silencing (46% GAPDH knockdown) exceeded levels achieved in the media/adventitia (8% pixels fluorescing, 24% GAPDH knockdown) across all transfection methods. CONCLUSION: Endothelial gene silencing is possible within the time frame and conditions of surgical application without the use of transfection reagents. The high sensitivity of endothelial cells to siRNA transfection marks the endothelium as a promising target of gene therapy in vascular disease.

Low ejection fraction predicts shortened survival in patients undergoing infrainguinal arterial reconstruction.

BACKGROUND: With the advent of endovascular therapy for lower extremity ischemia it is important to better determine what factors may affect the outcome. The goal of the present study was to evaluate whether ejection fraction (EF) is predictive of outcome in infrainguinal arterial reconstruction. METHODS: We retrospectively reviewed 736 patients undergoing 897 infrainguinal arterial reconstructions from July 1999 to February 2002. Patients were divided into two groups: group I contained 54 patients with an EF<35% and group II had 216 patients with an EF > or =35%. The outcome evaluated was major adverse clinical events (MACEs), defined as postoperative myocardial infarction (MI), arrhythmia, congestive heart failure (CHF), and perioperative mortality. RESULTS: Major adverse clinical events occurred in 20.3% of patients (11/54) in group I and 10.6% patients (23/216) in group II (p = 0.068). Group I had a trend toward a greater incidence of MACEs compared to group II. Two-year survival for group I was 61.7%, whereas survival for group II was 78.4% (p = 0.0085). CONCLUSIONS: Low EF predicts a significantly shortened 2-year survival after infrainguinal arterial reconstruction and a trend toward increased perioperative complications. This is another factor to be considered in choosing open versus endovascular options.

Lower extremity arterial revascularization in obese patients.

BACKGROUND: Obesity and associated comorbidities are associated with a high rate of complications and technical difficulties after a number of surgical procedures. We studied the role of obesity in outcomes in lower extremity arterial revascularization. METHODS: We reviewed all lower extremity arterial revascularizations performed at our institution in 2000. Body mass index (BMI) greater than or equal to 30 kg/m(2) defined obesity. Perioperative outcomes, long-term survival, and graft patency were evaluated in obese and nonobese patients by using linear regression, the Fisher exact test, and Kaplan-Meier analysis. RESULTS: The study population consisted of 74 (26%) obese and 207 (74%) nonobese patients. Patient demographics of the obese and nonobese populations were similar. The mean BMI for obese patients was 35 +/- 5 kg/m(2) and in nonobese patients was 25 +/- 3 kg/m(2). The mean age of each group was 67 +/- 10 years (BMI > or =30 kg/m(2)) and 70 +/- 13 years (BMI <30 kg/m(2)). There were 45 (61%) obese men and 29 (39%) obese women. There were 128 (62%) nonobese men and 79 (38%) nonobese women. Diabetes was present in 76% of the obese and 70% of the nonobese patients. Perioperative myocardial infarction, 30-day mortality, and rate of reoperation within 30 days were not significantly different. Obese patients had higher increased postoperative wound infection rates (16% vs 7%; P = .04). Survival analysis showed 81% +/- 5% and 85% +/- 3% 1-year survival and 66% +/- 6% and 62% +/- 3% 3-year survival in obese and nonobese patients (P = .58), respectively. Kaplan-Meier estimates showed no effect of obesity on long-term graft patency, with 1-year graft patency rates of 82% +/- 6% and 81% +/- 4% in obese and nonobese patients, respectively (P = .79). CONCLUSIONS: Obese patients have similar limb salvage rates, perioperative cardiac morbidity, long-term survival rates, and long-term graft patency but have increased perioperative wound infections.

A novel function for cadherin 11/osteoblast-cadherin in vascular smooth muscle cells: modulation of cell migration and proliferation.

OBJECTIVE: Intimal hyperplasia is a common cause of vein graft failure in cardiovascular surgery. The molecular basis for intimal hyperplasia remains poorly defined. We have previously identified, by gene chip analysis of vein grafts, increased messenger (mRNA) for the adhesion molecule cadherin 11/osteoblast-cadherin (CDH11). The function of CDH11 in vascular cells is unknown. The aim of the present study is to confirm CDH11 expression in vein grafts and characterize its role in vascular remodeling. METHODS: Cephalic vein interposition grafts were implanted in a canine model and harvested at predetermined time points. CDH11 protein expression was determined by immunohistochemistry. Early passage human coronary artery smooth muscle cells (SMCs) were used for in vitro studies. Real-time polymerase chain reaction was used to assess cellular CDH11 mRNA levels. CDH11 signaling was inhibited by either transfection with silencing RNA targeting CDH11 or with a blocking antibody to CDH11. Cellular migration was evaluated and cellular proliferation was assessed. RESULTS: Expression of CDH11 was increased in medial SMCs of vein grafts recovered at 7, 14, and 30 days after surgery compared with control veins from the same animals. In vitro CDH11 mRNA was up-regulated 1.8 +/- 0.2-fold (P = .003) in SMCs after treatment with tumor necrosis factor-alpha. Cellular migration was attenuated by inhibition of CDH11 both with a blocking antibody (0.67 +/- 0.09; P = .063) and gene knockdown mediated by small interfering RNA (0.67 +/- 0.14; P = .036). SMC proliferation decreased by 3.1-fold (P = .006) in the presence of CDH11-blocking antibody. Knockdown of CDH11 mediated by small interfering RNA resulted in a 1.3-fold (P = .018) decrease in proliferation. CONCLUSIONS: CDH11 is up-regulated in SMC in vivo and in vitro as part of the response to injury. Inhibition of CDH11 decreases SMC migration and proliferation, two pathogenic effectors of intimal hyperplasia.

Creatinine clearance but not serum creatinine alone predicts long-term postoperative survival after lower extremity revascularization.

BACKGROUND: Renal insufficiency is a well-described risk factor for perioperative morbidity and shortened survival after major vascular procedures. Due to the potential inaccuracy of serum creatinine levels alone in measuring kidney function, our aim was to determine whether estimated creatinine clearance more consistently predicted long-term survival. METHODS: A retrospective review of one institution's vascular registry was performed. Logistic regression analysis was conducted to determine independent predictors of 1-, 2- and 3-year postoperative mortality. Creatinine clearance was estimated as [140 - age (years)] x weight (kg)/72 x serum creatinine (mg/dl), multiplied by 0.85 for women. RESULTS: A total of 252 consecutive patients underwent infrainguinal bypass procedures between August 1999 and May 2000. Demographics included average age 68 years, 65% male, 74% diabetic, 12% dialysis-dependent, 23% history of congestive heart failure, 12% history of stroke and 20% serum creatinine >2 mg/dl. One-year mortality was 16% (n = 40), 2-year mortality was 25% (n = 64), and 3-year mortality was 35% (n = 88). There was no difference in serum creatinine values between survivors and non-survivors at 1 year (1.8 vs. 1.9, p = 0.80), 2 years (1.8 vs. 2.0, p = 0.62) or 3 years (1.8 vs. 2.0, p = 0.24), and creatinine >2 mg/dl did not predict long-term adverse outcomes. In contrast, reduced creatinine clearance (< or =60 ml/min) was an independent predictor of mortality regardless of dialysis status (1 year: OR = 2.53, p = 0.014; 2 years: OR = 2.46, p = 0.004; 3 years: OR = 2.45, p = 0.001), and creatinine clearance was higher for survivors versus non-survivors at all 3 time points (1 year: 70.2 vs. 49.5, p = 0.003; 2 years: 72.3 vs. 51.2, p < 0.0001; 3 years: 74.7 vs. 52.6, p < 0.0001). Other independent predictors of mortality included a history of stroke (1 year: OR = 3.28, p = 0.008; 2 years: OR = 2.55, p = 0.025; 3 years: OR = 2.35, p = 0.038) and congestive heart failure (1 year: OR = 2.86, p = 0.006; 2 years: OR = 2.54, p = 0.005; 3 years: OR = 2.13, p = 0.017). CONCLUSIONS: Independent of dialysis status, a decreased creatinine clearance, but not elevated serum creatinine alone, is an independent predictor of mortality after lower extremity arterial reconstruction. Determination of creatinine clearance should replace serum creatinine in the preoperative risk evaluations of patients undergoing major vascular surgical procedures.