Comparative analysis of polymers for short interfering RNA delivery in vascular smooth muscle cells.

UNLABELLED: The use of short interfering RNA (siRNA) to degrade messenger RNA in the cell cytoplasm and transiently attenuate intracellular proteins shows promise in the inhibition of vascular pathogenesis. However, a critical obstacle for therapeutic application is a safe and effective delivery system. Biodegradable polymers are promising alternative molecular carriers for genetic material. Here, we aim to perform a comparative analysis of poly(B-amino ester) (PBAE) and polyethylenimine (PEI) polymers in their efficacy for vascular smooth muscle cell transfection using siRNA against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene as our test target. METHODS: Human aortic smooth muscle cells (HASMC) were transfected in vitro with polymers conjugated to GAPDH or negative control (NC) siRNAs. Increasing siRNA:polymer ratios were tested for optimal transfection efficiency. DharmaFECT2 chemical transfection complexes were used for comparative analysis. Live/dead dual stain was used to measure cell viability, and GAPDH gene silencing was measured by quantitative polymerase chain reaction normalized to 18S. RESULTS: The highest rate of PEI-mediated silencing was achieved with a 9µL polymer:220 pmol/mL siRNA conjugate (16 ± 2% expression versus NC; n = 6). Comparable PBAE-mediated silencing could be achieved with a 1.95µL polymer:100 pmol/mL siRNA conjugate (10 ± 1% expression versus NC; n = 5). Transfection using PEIs resulted in silencing equivalent to other methods but with less efficiency and increased cell toxicity at 24h polymer exposure. Decreasing PEI exposure time to 4 h resulted in similar silencing efficacy (21 ± 9% expression versus NC, n = 6) with an improved toxicity profile. CONCLUSIONS: Polymeric bioconjugates transfected HASMCs in a manner similar to chemical complexes, with comparable cell toxicity and silencing efficiency. PEI bioconjugates demonstrated silencing equivalent to PBAE bioconjugates, although less efficient in terms of required polymer concentrations. Given the cost-to-benefit difference between the assayed polymers, and PEI's ability to transfect HASMCs within a short duration of exposure with an improved toxicity profile, this study shows that PEI bioconjugates are a potential transfection agent for vascular tissue. Future studies will expand on this method of gene therapy to validate delivery of gene-specific inhibitors aimed at attenuating smooth muscle cell proliferation, adhesion, and migration. These studies will lay the framework for our future experimental plans to expand on this method of gene therapy for in vivo transfection in animal models of vascular disease.

Early versus delayed amputation in the setting of severe lower extremity trauma.

Leg-threatening injuries present patients and clinicians with the difficult decision to pursue primary amputation or attempt limb salvage. The effects of delayed amputation after failed limb salvage on outcomes, such as prosthetic use and hospital deposition, are unclear. We evaluated the timing of amputations and its effects on outcomes. We retrospectively reviewed all trauma patients undergoing lower extremity amputation from January 1, 2000 through December 31, 2010 at a Level 2 trauma center. Patients undergoing early amputation (amputation within 48 hours of admission) were compared with patients undergoing late amputation (amputations >48 hours after admission). Patient demographics, injury specifics, operative characteristics, and outcomes were documented. During the 11-year study period, 43 patients had a lower extremity amputation and 21 had early amputations. The two groups were similar except for a slightly higher Mangled Extremity Severity Score in the early amputation group. Total hospital length of stay significantly differed between groups, with the late amputation group length of stay being nearly twice as long. The late amputation group had significantly more ipsilateral leg complications than the early group (77% vs 15%). There was a trend toward more prosthetic use in the early group (93%vs 57%, P = 0.07). Traumatic lower extremity injuries requiring amputation are rare at our institution (0.3% incidence). Regardless of the amputation timing, most patients were able to obtain a prosthetic. Although the late group had a longer length of hospital stay and more local limb complications, attempted limb salvage still appears to be a viable option for appropriately selected trauma patients.

The effect of temporal artery biopsy on the treatment of temporal arteritis.

BACKGROUND: Temporal artery biopsy (TAB) is frequently used to guide treatment for suspected temporal arteritis. Our purpose was to determine the influence on subsequent temporal arteritis treatment, particularly the initiation, termination, or continuation of corticosteroids after a histologically negative TAB. METHODS: This is a retrospective analysis from a single regional referral center on all patients undergoing TAB March 2003 through November 2010. Demographic, clinical, and surgical informations were recorded including changes in treatment based on biopsy results. RESULTS: In all, 237 patients had complete documentation for review; the average age was 71 years (range 34 to 94) and 56% were women. Thirty-six patients had 42 positive biopsies; 26 biopsies were bilateral. Positive biopsy results were defined as having marked intimal thickening, transmural inflammation, and "giant cells." Neither length of biopsy specimen nor preoperative steroid use affected pathologic diagnosis (2.41 vs 2.38 cm, P = .46, and 52% vs 50%, P = .8, respectively). Symptoms included new-onset headache (75%), preauricular tenderness and jaw claudication (32%), erythrocyte sedimentation rate greater than 50 mm/h (60%), and a score of 3 or more using the American College of Rheumatology criteria (56%). Among the 56% of patients who met the criteria for the clinical diagnosis of temporal arteritis, 22% demonstrated a positive biopsy. Biopsy results had no significant impact on subsequent treatment in 69% of patients who met clinical diagnostic criteria (P = .7); in the remaining 31%, biopsy results altered subsequent treatment with either corticosteroid initiation or discontinuation. CONCLUSIONS: The pathologic results of the TAB did not significantly affect treatment in most patients.