Wound healing is critically affected by age, ischemia, and growth factors such as TGFbeta1. The combined effect of these factors on fibroblast migration, an essential component of wound healing, is poorly understood. To address this deficiency, we examined expression of TGFbeta receptor type I and II (TGFbetaRI and RII) under normoxia or hypoxia (1% O(2)) in cultured human dermal fibroblasts (HDFs) from young (ages 24-33) and aged (ages 61-73) adults. TGFbetaRI and RII expression was similar in both groups under normoxia. Hypoxia did not alter receptor levels in young HDFs but significantly decreased TGFbetaRI in aged cells (12 and 43%, respectively). Additionally, young cells displayed a 50% increase in activation of p42/p44 mitogen-activated kinase by TGFbeta1 (2-200 pg/ml) under hypoxia while aged cell levels of active p42/p44 decreased up to 24%. To determine functional outcomes of these findings, we measured the migratory capacity of the cells on type I collagen using a gold salt migration assay. Hypoxia increased the migratory index (MI) of young HDFs over normoxia by 30% but had no effect on aged cells. Under normoxia, TGFbeta1 (1-1000 pg/ml) increased young HDF migration in a concentration-dependent manner up to 109% over controls but minimally increased aged HDF migration (37%). Under hypoxia, TGFbeta1 significantly increased young cell MI at all concentrations but was without effect on the aged HDF response. These data demonstrate that aged fibroblasts have an impaired migratory capacity with complete loss of responsiveness to hypoxia and deficits in the migratory and signal transduction responsiveness to TGFbeta1 that may partly explain diminished healing capabilities often observed in aged patients.

Homocysteine, a metabolite of the methionine cycle has been reported to play a role in neurotoxicity through activation of NMDAR-mediated signaling pathway. The proposed mechanisms associated with homocysteine-NMDAR induced neurotoxicity involve a unique signaling pathway that triggers a crosstalk between ERK and p38 MAPKs, where activation of p38 MAPK is downstream of and dependent on ERK MAPK. However, the molecular basis of the ERK MAPK mediated p38 MAPK activation is not understood. The current study investigates whether AMPARs play a role in facilitating the ERK MAPK mediated p38 MAPK activation. Using surface biotinylation and immunoblotting approaches we show that treatment with homocysteine leads to a decrease in surface expression of GluA2-AMPAR subunit in neurons, but has no effect on the surface expression of GluA1-AMPAR subunit. Inhibition of NMDAR activation with D-AP5 or ERK MAPK phosphorylation with PD98059 attenuates homocysteine-induced decrease in surface expression of GluA2-AMPAR subunit. The decrease in surface expression of GluA2-AMPAR subunit is associated with p38 MAPK phosphorylation, which is inhibited by NASPM, a selective antagonist of GluA2-lacking Ca2+-permeable AMPARs. These results suggest that homocysteine-NMDAR mediated ERK MAPK phosphorylation leads to a decrease in surface expression of GluA2-AMPAR subunit resulting in Ca2+ influx through the GluA2-lacking Ca2+-permeable AMPARs and p38 MAPK phosphorylation. Cell death assays further show that inhibition of AMPAR activity with NBQX/CNQX or GluA2-lacking Ca2+-permeable AMPAR activity with NASPM attenuates homocysteine-induced neurotoxicity. We have identified an important mechanism involved in homocysteine-induced neurotoxicity that highlights the intermediary role of GluA2-lacking Ca2+-permeable AMPARs in the crosstalk between ERK and p38 MAPKs.

Although tourniquets play an integral role in extremity surgery, no clear guidelines exist for the use of tourniquets in microsurgery. We undertook a study in 12 healthy volunteers to better understand the coagulation properties of blood distal to an inflated tourniquet. At a 15‐min inflation time, blood distal to an inflated tourniquet clots faster than blood taken from the opposite arm after addition of exogenous thrombin (12.5 s vs 17.5 s, P < 0.0001). Neither fibrinopeptide A (FPA) levels nor tissue plasminogen activator (tPA) levels were different from those of controls. Tissue factor pathway inhibitor (TFPI), an endogenous local anticoagulant, was slightly but significantly elevated in tourniquet blood. Although much remains to be understood, we believe that microvascular surgery in a bloodless field is safe and efficacious. Nine patients are presented who successfully underwent microvascular surgery in a bloodless field, using various types of extremity tourniquets. © 2000 Wiley‐Liss, Inc. MICROSURGERY 20:221–224 2000