Diana A. Lepore

Role of priming stresses and Hsp70 in protection from ischemia- reperfusion injury in cardiac and skeletal muscle

Abstract Ischemia-reperfusion injury limits the survival of muscle involved in tissue trauma or transfers during microsurgical reconstruction. Priming stresses such as ischemic preconditioning or mild hyperthermia have frequently been associated with improved survival of ischemic-reperfused cardiac muscle, such protection coinciding with induction of the stress-related heat shock protein 70 (Hsp70). Little is known about the response of skeletal muscle to priming stresses. This review summarizes the current knowledge on the use of priming stresses as protective strategies against the consequences of ischemia-reperfusion in cardiac and skeletal muscle and the potential role of Hsp70.

Prior heat stress improves survival of ischemic-reperfused skeletal muscle in vivo

The ability of heat stress to improve the survival of ischemic‐reperfused skeletal muscle in vivo was investigated. Ischemia‐reperfusion was applied using the rat hindlimb tourniquet model. The viability of ischemic‐reperfused muscle (11 ± 1%) was increased by prior mild heat stress (86 ± 2%). To investigate whether heat shock protein 70 (Hsp 70) expression in the muscle of the heated limb was responsible for this protection, the survival of Hsp 70–expressing transduced myoblasts and myocytes was measured after exposure to mediators of ischemia‐reperfusion injury. Survival was improved in Hsp 70–positive myoblasts but not in myocytes, suggesting that the mechanism of protection conferred by heat stress in vivo cannot be explained by the expression of Hsp 70 in myocytes and may involve a more complex mechanism. In conclusion, prior heat stress is effective in protecting mature skeletal muscle in vivo against necrosis after ischemia‐reperfusion and has potential for use in microsurgical procedures requiring tourniquet applications.

Survival and Differentiation of Pituitary Colony‐Forming Cells In Vivo

Growth hormone (GH) deficiency is a significant clinical problem, since growth hormone is essential for the regulation of growth, metabolism, and the cardiovascular system. Stem and progenitor cells have been identified in many adult tissues. Recently, our laboratory identified a cell type within the adult pituitary gland with stem cell‐like properties, which we have termed pituitary colony‐forming cells (PCFCs). Herein we investigate the ability of PCFCs to survive and differentiate in vivo. Enriched populations of PCFCs were transplanted into an in vivo microchamber model. Grafts were harvested at 6 weeks post‐transplant and tested for surviving donor cells (LacZ(+)) or for differentiation (GH(+)). The results showed that donor cells survived in chambers (LacZ(+)) and underwent division (phosphohistone‐H3‐positive). Furthermore, grafted cells showed colocalization of LacZ and GH, suggesting differentiation. To confirm differentiation, donor cells were obtained from a GH‐enhanced green fluorescent protein (eGFP) reporter transgenic mouse model that expressed eGFP under control of the GH promoter. Cells that were eGFP(−), that is, GH(−), were selected by fluorescence‐activated cell sorting (FACS) and transplanted. After 6 weeks, eGFP(+)GH(+) cells were detected in grafts by immunostaining and by FACS analysis of digested grafts. In conclusion, PCFCs have the capacity to divide and differentiate into GH(+) cells in vivo. The vascularized tissue chamber model is an ideal model to investigate the environmental niche for PCFC expansion and differentiation and has the potential to be developed into a growth hormone‐releasing organoid in vivo.

A Role for Angiotensin‐Converting Enzyme in the Characterization, Enrichment, and Proliferation Potential of Adult Murine Pituitary Colony‐Forming Cells

Recently, we described a rare cell type within the adult murine pituitary gland with progenitor cell hallmarks (PCFCs). PCFCs are contained exclusively within a subpopulation of cells that import fluorescent β‐Ala‐Lys‐Nε‐AMCA (7‐amino‐4‐methylcoumarin‐3‐acetic acid). Herein, we investigate the utility of cell surface molecules angiotensin‐converting enzyme (ACE) and stem cell antigen‐1 (Sca‐1) to further enrich for PCFCs. ACE and Sca‐1 were expressed on 61% and 55% of AMCA+CD45−CD31− cells, respectively, and coexpressed on 38%. ACE+Sca‐1+AMCA+ cells enriched for PCFCs by 195‐fold over unselected cells. ACE+AMCA+ cells enriched for PCFCs by 170‐fold, and colonies were twofold larger than for AMCA+ selection alone. Conversely, ACE−‐selected cells reduced both colony‐forming activity and size. Notably, colonies generated from AMCA+ cells obtained from ACEnull mice were 2.7‐fold smaller than for wild‐type mice. These data identify ACE as a previously unrecognized marker of PCFCs and suggest that ACE is functionally important for PCFC proliferation. Anatomically, the cells that imported AMCA and expressed ACE were situated in the marginal epithelial cell layer of the pituitary cleft and in the adjacent subluminal zone, thus supporting previous proposals that the luminal zone is a source of precursor cells in the adult pituitary.

Effects of the endothelin receptor antagonist Bosentan on ischaemia/reperfusion injury in rat skeletal muscle.

We examined the role of endothelin in ischaemia/reperfusion injury in skeletal muscle, using the endothelin receptor antagonist Bosentan. In the rat hindlimb tourniquet ischaemia model, one hindlimb was rendered ischaemic for 2 h at 36 degrees C, then blood flow was re-established for either 24 h to assess muscle survival or 1.5 h for a study of capillary perfusion. In the first set of rats, the gastrocnemius muscle was removed from the postischaemic limb and assessed for viability histochemically using the nitro blue tetrazolium stain. Tissue water content (a measure of oedema) and myeloperoxidase activity (a measure of neutrophil accumulation) were also assessed in the ischaemic muscle, the contralateral non-ischaemic muscle and the lungs. In the second set of rats, the hind limb was infused with India ink after 2-h ischaemia and 1.5-h reperfusion and the muscle was harvested, fixed and cleared. In control rats, muscle viability was 17+/-2% (S.E.M.). In rats treated with Bosentan (10 mg/kg, i.p.) 30 min before release of the tourniquet, muscle viability (48+/-7%) was significantly increased compared to the control group (P<0.01). Bosentan treatment had no significant effect on tissue water content or myeloperoxidase activity in the ischaemic muscle, the contralateral non-ischaemic muscle or the lung. Immunoreactive endothelin levels in serum increased to a peak at 90 min of reperfusion and returned to control levels by 24-h reperfusion. India ink studies demonstrated a significantly increased functional capillary density in postischaemic Bosentan-treated muscles compared with postischaemic control muscles (P<0.05). These results suggest that endothelin plays an important role in the necrosis which results from a period of ischaemia and reperfusion in skeletal muscle, by mediating a decrease in postischaemic microvascular perfusion.

Cool perfusion solutions for skin flaps: a new mixture of pharmacological agents which improves skin flap viability

This study tested the hypotheses that perfusion of cooled skin flaps with established organ preservation solutions improves their viability and that this improvement can be further enhanced by pharmacological manipulation. Rabbit epigastric skin flaps were perfused with different solutions before explantation and stored at 8 degrees C for 6 days. In the first part of the experiment, flap viability was assessed 7 days after reperfusion of the flap via microvascular anastomoses. The different solutions were heparinised blood, University of Wisconsin solution, two of its modifications, EuroCollins solution and a pharmacological mixture containing phosphoenolpyruvate, desferrioxamine, nitrendipine, dextran 70 and a platelet-aggregating factor receptor antagonist (WEB 2170). In the second part, biochemical parameters of skin were measured at various reperfusion times. Adenosine triphosphate (ATP), reduced glutathione (GSH), myeloperoxidase (MPO) and tissue water were assayed at 0, 1, and 24 h after reperfusion. In addition, plasma thromboxane (TXB2) was measured at 0, 30 and 60 minutes after reperfusion. The viability of flaps perfused with the mixture (81%) was significantly higher than that of any of the other groups (39% for controls, 38% for EuroCollins, 13% for UW solution, 27% and 31% for its modifications). ATP levels after reperfusion were higher in the mixture group than in UW-perfused group. GSH levels in the mixture group were also higher than in the UW group, indicating higher level of protection against oxidative stress during reperfusion. There were no differences in MPO levels. Thromboxane levels associated with UW-perfused flaps were significantly higher than those associated with any other perfusion solution. In conclusion, perfusion of a mixture of pharmacological agents targeting specific aspects of ischaemia/reperfusion injury improved the viability of skin flaps stored in the cold for 6 days, whereas standard organ preservation solutions failed to affect significantly skin flap survival.

Mild hypothermia protects against ischaemia-reperfusion injury in rabbit skeletal muscle

In three groups of rabbits, the rectus femoris muscle was subjected to 4 hours of total ischaemia. In Group 1 (normothermia, n = 5) the core temperature was maintained within the range 36-38 degrees C for the duration of ischaemia. In Group 2 (total hypothermia, n = 5) the core temperature was allowed to fall to 31.5-33.5 degrees C. In Group 3 (muscle only hypothermia, n = 5) core temperature was maintained as in Group 1 but the muscle temperature was allowed to fall to 29.5-31.5 degrees C. After 24 hours of reperfusion the muscles were harvested and measurements made of muscle viability, oedema and myeloperoxidase content. The mean (s.e.m.) muscle viability of Group 1, 19.5 (3.8)%, was significantly less than that of both Group 2, 86.0 (2.0)%, and Group 3, 87 (4.1)%, (P < 0.001). Muscle oedema and myeloperoxidase levels were elevated in all experimental groups, but differences were not significant. These findings indicate that ischaemia-reperfusion injury in skeletal muscle in this model is highly temperature-sensitive, small reductions in muscle temperature during ischaemia providing significant protection against ischaemia-reperfusion injury.

Interaction between thromboxane and free radical mechanisms in experimental ischaemic rabbit skin flaps.

SummaryThe possible relationship between increased blood levels of thromboxane (TXA2) and tissue levels of free radicals during ischaemia was investigated. Rabbit epigastric skin flaps were subjected to 4h of body temperature ischaemia, then infused with either the TXA2 synthetase inhibitor UK-38,485, the free radical scavenger superoxide dismutase (SOD), or both immediately prior to reperfusion. After 30 min of reperfusion, increases in the tissue levels of xanthine oxidase (XO) and malonyldialdehyde (MDA), both of which are indices of free radical generation and decreases in the tissue levels of SOD were found. SOD treatment completely restored XO, MDA and SOD levels to normal, whereas UK-38,485 only partially improved all three parameters. None of these changes was statistically significant. Effluent blood thromboxane B2 (TXB2) levels from the flap increased significantly (P<0.01) after ischaemia and were reduced significantly by both UK-38,485 and SOD (P<0.05). Combined UK-38,485 and SOD treatment was no better than treatment with either agent alone. ATP levels and oedema, which decreased and increased respectively due to ischaemia, were not significantly altered by drug infusion. These results suggest that free radical damage may be related to TXA2-generated thrombosis in ischaemia/reperfusion injury.

Glycosaminoglycan composition of uninjured skin and of scar tissue in fetal, newborn and adult sheep.

Few details are available on the heterogeneity of glycosaminoglycans (GAGs) in healing fetal wound tissue. We used a sensitive assay for hexosamines to examine changes occurring in the development of normal sheep skin and of wound healing tissue in PVA sponges inserted subcutaneously at different stages of gestation. It was assumed that glucosamine was derived mainly from hyaluronan and galactosamine mainly from dermatan sulphate and chondroitin sulphate. Hexosamine-containing tissue infiltrating the sponges was deposited more repidly in the first week than in the second week. Three days after wounding, approximately 70% of the total GAGs in wound tissue was hyaluronan. The proportion of hyaluronan then fell progressively and by the 14th day contributed 57% to total GAGs. In uninjured skin the contribution of hyaluronan to the total GAGs fell progressively with increasing fetal maturity, the level being 70% at 75 days gestation, but only 35–40% in newborn or adult skin. At no stage of development was there a sudden change in GAG composition suggestive of a transition from regeneration to scar formation. It is concluded that hyaluronan may play an important role in the biochemical sequence leading to collagen fibrillogenesis and mature scar formation.

Differentiation of mouse embryonic stem cells into growth hormone and prolactin expressing cells in vitro.

Embryonic stem (ES) cells provide an excellent in vitro model system to investigate the cellular and molecular events that drive lineage-specific commitment and differentiation and have considerable potential as agents for novel cell-based therapies. While culture methods for directed in vitro differentiation of ES into neuroectodermal and mesodermal derivatives are relatively well established, little is known about the capacity of ES cells to generate the hormone secreting lineages of the pituitary. Here we show that extended culture of adherent embryoid bodies (EBs) results in the induction of pituitary-specific progenitor marker genes and pituitary hormonal markers. GH-expressing cells comprise approximately 2% of the total viable cell population and exhibit dense granular cytoplasmic immunoreactivity. These data demonstrate for the first time that cell types which have the characteristics of pituitary somatotropes and lactotropes can be generated by in vitro differentiation of mouse ES cells.