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

BackgroundImpaired 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.MethodsDMVECs 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.ResultsAs 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.ConclusionHyperglycemic 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.Clinical RelevanceChronic diabetic foot ulceration, as a result of impaired wound healing in diabetes, is the source of a vast quantity of hospitalizations and lower extremity amputations across the nation. Normal wound healing involves the bidirectional signaling and interaction between a multitude of inflammatory cytokines and neuropeptides, and previous research has demonstrated a dysregulation in the expression and activity of these molecules in diabetes. This work evaluates the effect of hyperglycemic conditions and neuropeptides on DMVECs, a cell type fundamental to the wound healing process, but in which research is currently limited. The discovery of a molecule, which promotes angiogenesis in hyperglycemic conditions would be a promising therapeutic target for impaired wound healing in diabetes. In addition, the determination of the effects of these neuropeptides and cytokines on DMVECs could elucidate other potential therapies, which could decrease wound-healing time and improve overall wound healing in other realms of medicine

RNAi therapy to the wall of arteries and veins: anatomical, physiologic, and pharmacological considerations

Background: Cardiovascular disease remains a major health care challenge. The knowledge about the underlying mechanisms of the respective vascular disease etiologies has greatly expanded over the last decades. This includes the contribution of microRNAs, endogenous non-coding RNA molecules, known to vastly influence gene expression. In addition, short interference RNA has been established as a mechanism to temporarily affect gene expression. This review discusses challenges relating to the design of a RNA interference therapy strategy for the modulation of vascular disease. Despite advances in medical and surgical therapies, atherosclerosis (ATH), aortic aneurysms (AA) are still associated with high morbidity and mortality. In addition, intimal hyperplasia (IH) remains a leading cause of late vein and prosthetic bypass graft failure. Pathomechanisms of all three entities include activation of endothelial cells (EC) and dedifferentiation of vascular smooth muscle cells (VSMC). RNA interference represents a promising technology that may be utilized to silence genes contributing to ATH, AA or IH. Successful RNAi delivery to the vessel wall faces multiple obstacles. These include the challenge of cell specific, targeted delivery of RNAi, anatomical barriers such as basal membrane, elastic laminae in arterial walls, multiple layers of VSMC, as well as adventitial tissues. Another major decision point is the route of delivery and potential methods of transfection. A plethora of transfection reagents and adjuncts have been described with varying efficacies and side effects. Timing and duration of RNAi therapy as well as target gene choice are further relevant aspects that need to be addressed in a temporo-spatial fashion. Conclusions: While multiple preclinical studies reported encouraging results of RNAi delivery to the vascular wall, it remains to be seen if a single target can be sufficient to the achieve clinically desirable changes in the injured vascular wall in humans. It might be necessary to achieve simultaneous and/or sequential silencing of multiple, synergistically acting target genes. Some advances in cell specific RNAi delivery have been made, but a reliable vascular cell specific transfection strategy is still missing. Also, off-target effects of RNAi and unwanted effects of transfection agents on gene expression are challenges to be addressed. Close collaborative efforts between clinicians, geneticists, biologists, and chemical and medical engineers will be needed to provide tailored therapeutics for the various types of vascular diseases

Efficacy of dorsal pedal artery bypass in limb salvage for ischemic heel ulcers

AbstractPurpose: Although pedal artery bypass has been established as an effective and durable limb salvage procedure, the utility of these bypass grafts in limb salvage, specifically for the difficult problem of heel ulceration, remains undefined. Methods: We retrospectively reviewed 432 pedal bypass grafts placed for indications of ischemic gangrene or ulceration isolated to either the forefoot (n = 336) or heel (n = 96). Lesion-healing rates and life-table analysis of survival, patency, and limb salvage were compared for forefoot versus heel lesions. Preoperative angiograms were reviewed to evaluate the influence of an intact pedal arch on heel lesion healing. Results: Complete healing rates for forefoot and heel lesions were similar (90.5% vs 86.5%, P = .26), with comparable rates of major lower extremity amputation (9.8% vs 9.3%, P = .87). Time to complete healing in the heel lesion group ranged from 13 to 716 days, with a mean of 139 days. Preoperative angiography demonstrated an intact pedal arch in 48.8% of the patients with heel lesions. Healing and graft patency rates in these patients with heel lesions were independent of the presence of an intact arch, with healing rates of 90.2% and 83.7% (P = .38) and 2-year patency rates of 73.4% and 67.0% in complete and incomplete pedal arches, respectively. Comparison of 5-year primary and secondary patency rates between the forefoot and heel lesion groups were essentially identical, with primary rates of 56.9% versus 62.1% (P = .57) and secondary rates of 67.2% versus 60.3% (P = .50), respectively. Conclusion: Bypass grafts to the dorsalis pedis artery provide substantial perfusion to the posterior foot such that the resulting limb salvage and healing rates for revascularized heel lesions is excellent and comparable with those observed for ischemic forefoot pathology. (J Vasc Surg 1999;30:499-508.

The use of arm vein in lower-extremity revascularization: Results of 520 procedures performed in eight years

AbstractPurpose: The absence of an adequate ipsilateral saphenous vein in patients requiring lower-extremity revascularization poses a difficult clinical dilemma. This study examined the results of the use of autogenous arm vein bypass grafts in these patients. Methods: Five hundred twenty lower-extremity revascularization procedures performed between 1990 and 1998 were followed prospectively with a computerized vascular registry. The arm vein conduit was prepared by using intraoperative angioscopy for valve lysis and identification of luminal abnormalities in 44.8% of cases. Results: Seventy-two (13.8%) femoropopliteal, 174 (33.5%) femorotibial, 29 (5.6%) femoropedal, 101 (19.4%) popliteo-tibial/pedal, and 144 (27.7%) extension “jump” graft bypass procedures were performed for limb salvage (98.2%) or disabling claudication (1.8%). The average age of patients was 68.5 years (range, 32 to 91 years); 63.1% of patients were men, and 36.9% of patients were women. Eighty-five percent of patients had diabetes mellitus, and 77% of patients had a recent history of smoking. The grafts were composed of a single arm vein segment in 363 cases (69.8%) and of spliced composite vein with venovenostomy in 157 cases (30.2%). The mean follow-up period was 24.9 months (range, 1 month to 7.4 years). Overall patency and limb salvage rates for all graft types were: primary patency, 30-day = 97.0% ± 0.7%, 1-year = 80.2% ± 2.1%, 3-year = 68.9% ± 3.6%, 5-year = 54.5% ± 6.6%; secondary patency, 30-day = 97.0% ± 0.7%, 1-year = 80.7% ± 2.1%, 3-year = 70.3% ± 3.4%, 5-year = 57.5% ± 6.2%; limb salvage, 30-day = 97.6% ± 0.7%, 1-year = 89.8% ± 1.7%, 3-year = 82.1% ± 3.3%, 5-year = 71.5% ± 6.9%. Secondary patency and limb salvage rates were greatest at 5 years for femoropopliteal grafts (69.8% ± 12.8%, 80.7% ± 11.8%), as compared with femorotibial (59.6% ± 10.3%, 72.7% ± 10.5%), femoropedal (54.9% ± 25.7%, 56.8% ± 26.9%,) and popliteo-tibial/pedal grafts (39.0% ± 7.3%, 47.6% ± 15.4%). The patency rate of composite vein grafts was equal to that of single-vein conduits. The overall survival rate was 54% at 4 years. Conclusion: Autogenous arm vein has been used successfully in a wide variety of lower-extremity revascularization procedures and has achieved excellent long- and short-term patency and limb salvage rates, higher than those generally reported for prosthetic or cryopreserved grafts. Its durability and easy accessibility make it an alternative conduit of choice when an adequate saphenous vein is not available. (J Vasc Surg 2000;31:50-9.

The effects of transfection reagent polyethyleneimine (PEI) and non-targeting control siRNAs on global gene expression in human aortic smooth muscle cells

Background: RNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a “noncoding RNAi”. However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen). Results: Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA Binding Protein (GATA)-4 and the methylation enzyme, Enhancer of zeste homolog 2 (EZH-2) a cytokine with an apical role in initiating the inflammatory response. Conclusions: Significant off-target effects in HAoSMCs transfected with PEI alone or in combination with control siRNAs may lead to misleading conclusions concerning the effectiveness of a targeted siRNA strategy. The lack of structural information about transfection reagents and “non coding” siRNA is a hindrance in the development of siRNA based therapeutics. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2267-9) contains supplementary material, which is available to authorized users

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

Cutaneous microcirculation in the neuropathic diabetic foot improves significantly but not completely after successful lower extremity revascularization

AbstractObjective: The purpose of this study was the examination of the effect of successful large vessel revascularization on the microcirculation of the neuroischemic diabetic foot. Research design and methods: We measured the cutaneous microvascular reactivity in the foot in 13 patients with diabetes with peripheral arterial disease and neuropathy (group DI) before and 4 to 6 weeks after successful lower extremity arterial revascularization. We also compared them with age-matched and sex-matched groups of 15 patients with diabetes and neuropathy, seven patients without neuropathy, and 12 healthy patients for control. We used single-point and laser Doppler scan imaging for the measurement of the foot skin vasodilatation in response to heating to 44°C and to iontophoresis of 1% acetylcholine (endothelial-dependent response) and 1% sodium nitroprusside (endothelial-independent response). Results: The group DI response to heat increased from 289% ± 90% before surgery (percent increase over baseline measured in volts) to 427% ± 61% (P <.05) after surgery but was still comparable with the response of the patients with diabetes and neuropathy (318% ± 51%) and lower than the responses of the patients without neuropathy (766% ± 220%) and the healthy patients for control (891% ± 121%; P <.0001). The group DI acetylcholine response also improved from 6% ± 4% before surgery to 26% ± 8% after surgery (P <.05) and was similar to the responses of patients with diabetes and neuropathy (18% ± 3%) and patients without neuropathy (38% ± 8%) but still lower when compared with the response of the patients for control (48% ± 9%; P <.001). The sodium nitroprusside response for group DI improved from 10% ± 4% to 29% ± 9% (P <.05) and was similar to the responses of the neuropathic (25% ± 9%), nonneuropathic (32% ± 6%), and control (40% ± 5%) groups. The group DI neurovascular response, which depends on the healthy function of the C-fiber nociceptors, was similar at baseline (5% ± 9%) and after surgery (14% ± %10) and in the neuropathic group (33% ± 21%), but it was dramatically reduced when compared with the nonneuropathic (110% ± 40%) and control (198% ± 54%) groups (P <.001). Conclusion: Impaired vasodilation in the diabetic neuropathic lower extremity leads to functional ischemia, which improves considerably but is not completely corrected with successful bypass grafting surgery. Therefore, patients with diabetes and neuropathy may still be at high risk for the development of foot ulceration or the failure to have an existing ulcer heal despite adequate correction of large vessel blood flow. (J Vasc Surg 2002;35:501-5.

High throughput RNAi assay optimization using adherent cell cytometry

<p>Abstract</p> <p>Background</p> <p>siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC).</p> <p>Methods</p> <p>AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo^®was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell.</p> <p>Results</p> <p>After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs.</p> <p>Conclusion</p> <p>This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate <it>in vitro </it>cell assays and thus save costs.</p