PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing.

BACKGROUND: Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing. METHODS: Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics. RESULTS: PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05). CONCLUSIONS: Silencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

Estimation of brachial artery volume flow by duplex ultrasound imaging predicts dialysis access maturation.

OBJECTIVE: This study validated duplex ultrasound measurement of brachial artery volume flow (VF) as predictor of dialysis access flow maturation and successful hemodialysis. METHODS: Duplex ultrasound was used to image upper extremity dialysis access anatomy and estimate access VF within 1 to 2 weeks of the procedure. Correlation of brachial artery VF with dialysis access conduit VF was performed using a standardized duplex testing protocol in 75 patients. The hemodynamic data were used to develop brachial artery flow velocity criteria (peak systolic velocity and end-diastolic velocity) predictive of three VF categories: low (<600 mL/min), acceptable (600-800 mL/min), or high (>800 mL/min). Brachial artery VF was then measured in 148 patients after a primary (n = 86) or revised (n = 62) upper extremity dialysis access procedure, and the VF category correlated with access maturation or need for revision before hemodialysis usage. Access maturation was conferred when brachial artery VF was >600 mL/min and conduit imaging indicated successful cannulation based on anatomic criteria of conduit diameter >5 mm and skin depth <6 mm. RESULTS: Measurements of VF from the brachial artery and access conduit demonstrated a high degree of correlation (R(2) = 0.805) for autogenous vein (n = 45; R(2) = 0.87) and bridge graft (n = 30; R(2) = 0.78) dialysis accesses. Access VF of >800 mL/min was predicted when the brachial artery lumen diameter was >4.5 mm, peak systolic velocity was >150 cm/s, and the diastolic-to-systolic velocity ratio was >0.4. Brachial artery velocity spectra indicating VF <800 mL/min was associated (P < .0001) with failure of access maturation. Revision was required in 15 of 21 (71%) accesses with a VF of <600 mL/min, 4 of 40 accesses (10%) with aVF of 600 to 800 mL/min, and 2 of 87 accesses (2.3%) with an initial VF of >800 mL/min. Duplex testing to estimate brachial artery VF and assess the conduit for ease of cannulation can be performed in 5 minutes during the initial postoperative vascular clinic evaluation. CONCLUSIONS: Estimation of brachial artery VF using the duplex ultrasound, termed the "Fast, 5-min Dialysis Duplex Scan," facilitates patient evaluation after new or revised upper extremity dialysis access procedures. Brachial artery VF correlates with access VF measurements and has the advantage of being easier to perform and applicable for forearm and also arm dialysis access. When brachial artery velocity spectra criteria confirm a VF >800 mL/min, flow maturation and successful hemodialysis are predicted if anatomic criteria for conduit cannulation are also present.

Management of spontaneous isolated visceral artery dissection.

BACKGROUND: Spontaneous isolated dissection of the celiac artery (CA) or the superior mesenteric artery (SMA) is rare but increasingly recognized because of widespread use of advanced abdominal imaging technology. Indications for specific therapeutic options and long-term outcomes are not well defined. This study analyzed clinical features, management strategies, and outcomes for patients with spontaneous visceral artery dissections. METHODS: Medical records of all patients diagnosed with CA or SMA dissections at 2 institutions (Scripps Green Hospital and San Diego Kaiser Medical Center) between January 2005 and January 2014 were retrospectively reviewed. Patient demographics included age, symptoms, associated comorbidities, and type of intervention. Anatomic features including length of dissection and entry point were measured. Efficacy of the various treatments was compared on the basis of symptom resolution and clinical course. RESULTS: Over an 8-year period, 23 patients with a diagnosis of visceral artery dissection were identified. Eighteen (78%) patients were men. Most patients (78%) were symptomatic on initial presentation with abdominal or back pain. Treatment included observation in 4, anticoagulation in 13, and endovascular stenting in 6 patients. The mean follow-up was 23.8 months. No patient required bowel resection. Twenty of twenty-three patients reported resolution of symptoms at follow-up. CONCLUSIONS: In this series, all patients with isolated visceral artery dissection had favorable outcomes, with no significant morbidity or mortality. Conservative management with anticoagulation is recommended as the first-line therapy. When conservative management fails, endovascular therapy is the treatment of choice.

Therapeutic angiogenesis for critical limb ischemia.

The application of gene- and cell-based therapies to promote angiogenesis is a novel concept to treat lower-limb critical limb ischemia (CLI) and may provide an unmet need for patients with no options for revascularization. Proof of concept was demonstrated in animal models resulting in clinical trials that have confirmed the feasibility and short-term efficacy of intramuscular injection of angiogenetic tissue growth factors or bone marrow stem cells. The safety of these biologic therapies has been demonstrated in randomized clinical trials with no "off-target" angiogenesis, growth of occult tumors, or progression of diabetic retinopathy. Current phase III randomized clinical trials using a DNA plasmid with the hepatocyte growth factor gene or bone marrow aspirate concentrate of mesenchymal cells are designed to address several crucial issues, including proper patient selection criteria, relevant clinical endpoints, and long-term efficacy. Because effectiveness of these novel therapies remains to be established, ongoing and future randomized clinical trials should be placebo-controlled, investigator-blinded, and have amputation-free survival as the primary endpoint. Further development of efficient gene transfer techniques and keeping transplanted stem cells healthy have the potential to make biologic therapies more robust in promoting angiogenesis, tissue regeneration, and resolution of CLI symptoms. If sustained efficacy can be demonstrated, new therapeutic strategies for patients with CLI will be available for clinicians, ie, limb revascularization using angiogenic gene or stem cell therapy alone, or in conjunction with endovascular intervention.

Interpretation and significance of ankle-brachial systolic pressure index.

The ankle-brachial systolic pressure index (ABI) is an underutilized, easy-to-perform, physiologic test to diagnosis atherosclerotic lower-limb arterial occlusive disease. Testing requires a sphygmomanometer and continuous-wave Doppler probe to measure the ratio of ankle and brachial systolic blood pressures. The ABI measurement has been standardized by use of the highest ankle and brachial systolic pressure for the calculation; abnormal threshold value is ≤0.9. The ABI is used to diagnosis and screen for peripheral arterial disease, which is a surrogate marker of atherosclerosis and a predictor of cardiovascular events. A lower ABI value correlates with severity of limb ischemia and decreased survival. Measurement of ABI is recommended as the initial diagnostic test for patients with exertional leg pain (claudication), to assess the healing potential of foot lesions, after blunt extremity trauma to detect occult arterial injury, and as part of the routine health assessment of patients with diabetes and peripheral arterial disease.

Adipose-derived stromal cells overexpressing vascular endothelial growth factor accelerate mouse excisional wound healing.

Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (ß-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

Antimycotic ciclopirox olamine in the diabetic environment promotes angiogenesis and enhances wound healing.

Diabetic wounds remain a major medical challenge with often disappointing outcomes despite the best available care. An impaired response to tissue hypoxia and insufficient angiogenesis are major factors responsible for poor healing in diabetic wounds. Here we show that the antimycotic drug ciclopirox olamine (CPX) can induce therapeutic angiogenesis in diabetic wounds. Treatment with CPX in vitro led to upregulation of multiple angiogenic genes and increased availability of HIF-1α. Using an excisional wound splinting model in diabetic mice, we showed that serial topical treatment with CPX enhanced wound healing compared to vehicle control treatment, with significantly accelerated wound closure, increased angiogenesis, and increased dermal cellularity. These findings offer a promising new topical pharmacologic therapy for the treatment of diabetic wounds.

The role of stem cells in cutaneous wound healing: what do we really know?

Wound repair is a complex process involving the orchestrated interaction of multiple growth factors, cytokines, chemokines, and cell types. Dysregulation of this process leads to problems such as excessive healing in the form of keloids and hypertrophic scars and chronic, nonhealing wounds. These issues have broad global implications. Stem cells offer enormous potential for enhancing tissue repair and regeneration following injury. The rapidly developing fields of stem cell biology and skin tissue engineering create translational opportunities for the development of novel stem cell-based wound-healing therapies.

Stem cells.

Stem cells are self-renewing cells capable of differentiating into multiple cell lines and are classified according to their origin and their ability to differentiate. Enormous potential exists in use of stem cells for regenerative medicine. To produce effective stem cell-based treatments for a range of diseases, an improved understanding of stem cell biology and better control over stem cell fate are necessary. In addition, the barriers to clinical translation, such as potential oncologic properties of stem cells, need to be addressed. With renewed government support and continued refinement of current stem cell methodologies, the future of stem cell research is exciting and promises to provide novel reconstructive options for patients and surgeons limited by traditional paradigms.

Institutional protocol improves retrievable inferior vena cava filter recovery rate.

BACKGROUND: In the trauma population, the use of retrievable inferior vena cava filters (RIVCF) is rapidly gaining acceptance in patients at high risk for venous thromboembolism. This study reports the impact of an institutional protocol on retrieval rates of RIVCF at a level I trauma center. METHODS: A review of an institutional Trauma Registry identified 94 consecutive patients who received RIVCF between January 2004 and February 2007 (group I) before the protocol was instituted. Under the protocol, 61 consecutive trauma patients received RIVCF between August 2007 and July 2008 (group II) and were prospectively followed. RESULTS: Filter retrieval eligibility criteria were met in 81% (76/94) of patients in group I and in 61% (37/61) of patients in group II. Of those eligible, retrieval-attempt rates were 42% (32/76) in group I versus 95% (35/37) in group II (P < .001). Clinician oversight of the filter accounted for 89% (39/44) of failure of retrieval attempts; patient noncompliance accounted for the rest in group I. In group II, the latter accounted for all such failures. Retrieval was successful in 37% (28/76) and in 84% (31/37) of the eligible patients in groups I and II, respectively (P < .001). No retrieval procedure-related complications occurred. CONCLUSION: An institutional protocol for prospective monitoring of RIVCF significantly increases filter retrieval rate.

Angiography for blunt splenic trauma does not improve the success rate of nonoperative management.

BACKGROUND: Splenic artery arteriography with possible therapeutic embolization (SAE) has been postulated to improve the success rate of nonoperative management of blunt splenic injuries and increase splenic salvage. Previous reports, however, have compared SAE with historical controls. We compared nonoperative success with SAE with a contemporaneous group treated nonoperatively without SAE. METHODS: Patients who suffered blunt splenic trauma from 2000 to 2004 were identified. Demographic and outcome data were abstracted. Data on the performance of SAE, type of vessel embolized, and success or failure of nonoperative management were collected. Analysis of variance, chi, and regression analysis were used to evaluate the impact of SAE on outcome. RESULTS: There were 570 patients who suffered blunt splenic trauma and 221 (39%) were treated operatively. There were 349 patients who were treated nonoperatively and 46 (13.2%) underwent SAE. SAE was more frequently used for patients with spleen Abbreviated Injury Score (AIS) > or =3 (31%) than AIS = 2 (6.7%). For patients with spleen AIS > or =3, there was no difference in age, gender, Injury Severity Score, or admission blood pressure between those who did or did not undergo SAE. The nonoperative success rate was similar for patients who did (79.3%) and those that did not (78.8%) undergo SAE. CONCLUSIONS: Patients who underwent splenic arteriography did not have improved nonoperative splenic salvage rates compared with a contemporaneous control group of similarly injured patients. Subsets of patients with blunt trauma may benefit from SAE but further study will be required to define these patients.

Modulation of chemotherapy resistance in regional therapy: a novel therapeutic approach to advanced extremity melanoma using intra-arterial temozolomide in combination with systemic O6-benzylguanine.

This study investigated whether the therapeutic index of regional melanoma therapy using parenteral temozolomide could be improved by chemomodulation with O6-benzylguanine (O6BG), an inhibitor of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Using a nude rat s.c. human melanoma xenograft model of the extremity, tumors were analyzed for AGT level 2 to 3 hours after the i.p. injection of 3.5 to 70.0 mg/kg O6BG to inhibit AGT activity. Survival studies were conducted using animals that were treated with a 15-minute isolated limb infusion with 10% DMSO in PBS (control), temozolomide alone, or temozolomide in conjunction with single or multiple doses of i.p. O6BG. Tumor volume and toxicity level were monitored every other day. Administration of 3.5 mg/kg O6BG depleted tumor AGT activity by 93.5% (P < 0.01). Groups treated with regional temozolomide alone (350 mg/kg), systemic temozolomide with O6BG, or vehicle combined with O6BG showed no significant tumor responses compared with controls. Whereas use of regional temozolomide alone at a higher dose (750 mg/kg) showed some degree of tumor response, regional temozolomide given in conjunction with multiple dosages of O6BG showed a marked (P < 0.01) reduction in tumor growth with minimal toxicity. Our findings suggest that AGT modulation by the administration of O6BG in combination with temozolomide regional chemotherapy leads to a significant improvement in melanoma antitumor responses. Clinical trials using chemotherapy modulation may improve response rates in future regional infusion and perfusion drug trials.

Optimizing a novel regional chemotherapeutic agent against melanoma: hyperthermia-induced enhancement of temozolomide cytotoxicity.

PURPOSE: Previous preclinical studies have shown that regional temozolomide therapy via isolated limb infusion is more effective than melphalan, the current drug of choice for regional chemotherapy for advanced extremity melanoma. The aim of this study was to determine whether hyperthermia could further augment the efficacy of temozolomide, an alkylating agent, against melanoma and improve its therapeutic index in a rat model of isolated limb infusion. EXPERIMENTAL DESIGN: Athymic rats bearing s.c. human melanoma xenografts (DM6) in their hind limbs were randomized to a 15-minute isolated limb infusion procedure with or without temozolomide at room temperature, normothermic (37.5 degrees C), or hyperthermic (43 degrees C) conditions. RESULTS: The concomitant administration of hyperthermia during an infusion with temozolomide led to the greatest increase in tumor growth delay, decreased proliferative index, and increased cell death. Isolated limb infusion treatment with a low dose (350 mg/kg) of temozolomide was ineffective at producing tumor growth delay (P = 0.07). Similarly, temozolomide infusion under normothermia yielded minimal tumor growth delay (P = 0.08). In contrast, the combination of hyperthermia plus temozolomide treatment produced marked tumor growth delay of 10.4 days (P = 0.02) with minimal toxicity. The addition of heat to temozolomide treatment yielded the smallest proliferative index (P = 0.001), while markedly increasing the level of apoptosis 48 hours after isolated limb infusion. CONCLUSION: This study, the first to examine the interaction between hyperthermia and temozolomide, shows a strong, synergistic antitumor effect when hyperthermia is combined with temozolomide for regional treatment of melanoma confined to an extremity. The mechanism of this synergy seems to be through an augmentation, by hyperthermia, of the antiproliferative and proapoptotic effects of temozolomide.

Temozolomide is a novel regional infusion agent for the treatment of advanced extremity melanoma.

BACKGROUND: Regional infusion therapy with melphalan (LPAM) is an accepted treatment for advanced extremity melanoma. However, much room exists for improving the therapeutic index of this type of therapy. METHODS: Isolated limb infusion (ILI) with temozolomide (TMZ), a novel methylating agent, was performed using a nude rat bearing human melanoma xenograft. Additional rats were treated systemically with TMZ, or regionally with LPAM or 10% dimethyl sulfoxide (DMSO; control) using ILI. RESULTS: Rats that received systemic TMZ showed a poor tumor response and no tumor regression. In contrast, intra-arterial TMZ demonstrated a prolongation of tumor growth delay in a dose-responsive manner. In comparison with LPAM of equitoxic dose, TMZ provided both longer tumor growth delay and a greater number of tumor regressions. CONCLUSIONS: These data suggest that ILI with TMZ is an effective treatment for advanced extremity melanoma and may be better than LPAM in this setting.