Periconception and First Trimester Diet Modifies Appetite, Hypothalamic Gene Expression, and Carcass Traits in Bulls.

Nulliparous yearling beef heifers (n=360) were used to evaluate the effects of maternal dietary protein during the periconception and first trimester periods of gestation on postnatal growth, feedlot performance, carcass characteristics, and the expression of genes associated with appetite in the arcuate nucleus of their male progeny. Heifers were individually fed a diet of 1.18g crude protein (CP)/day High protein (HPeri) or 0.62g CP/day Low protein (LPeri) beginning 60days before conception. From 24 to 98days post-conception (dpc), half of each treatment group changed to the alternative post-conception diet and were fed 1.49g CP/day (HPost) or 0.88g CP/day (LPost) yielding four treatment groups in a 2×2 factorial design. From day 98 of gestation, heifers received a common diet until parturition. Calves were weaned at 183days and developed on pasture before feedlot entry. Bulls underwent a 70-day Residual Feed Intake (RFI) feedlot test commencing at 528days of age. Feedlot entry and final body weight (BW), feedlot average daily gain (ADG) and RFI were not different (p>0.05). Progeny of dams that had a change in diet (LPeri/HPost and HPeri/LPost) had 9% higher daily dry matter intake (DMI) during the RFI test (p<0.05) than progeny of dams that received low diet throughout both the peri-conception period and first trimester (LPeri/LPost). Further, mRNA expression of the appetite-stimulating agouti-related protein (AGRP) was increased in the arcuate nucleus of High Peri/LPost bulls (p<0.05). Longissimus dorsi muscle cross sectional area, carcass dressing percentage, and estimated retail beef yield (RBY) were all higher (p<0.05), and rump (P8) fat tended to be lower (p=0.07), for bulls from HPost dams despite no difference in carcass weight (p<0.05). This study is of commercial importance to the livestock industry as specific periods of maternal dietary supplementation may increase feed intake, enhance progeny muscling, and alter fat deposition leading to improvement in efficiency of meat production in beef cattle.

Extreme drought pushes stream invertebrate communities over functional thresholds.

Functional traits are increasingly being used to predict extinction risks and range shifts under long-term climate change scenarios, but have rarely been used to study vulnerability to extreme climatic events, such as supraseasonal droughts. In streams, drought intensification can cross thresholds of habitat loss, where marginal changes in environmental conditions trigger disproportionate biotic responses. However, these thresholds have been studied only from a structural perspective, and the existence of functional nonlinearity remains unknown. We explored trends in invertebrate community functional traits along a gradient of drought intensity, simulated over 18 months, using mesocosms analogous to lowland headwater streams. We modelled the responses of 16 traits based on a priori predictions of trait filtering by drought, and also examined the responses of trait profile groups (TPGs) identified via hierarchical cluster analysis. As responses to drought intensification were both linear and nonlinear, generalized additive models (GAMs) were chosen to model response curves, with the slopes of fitted splines used to detect functional thresholds during drought. Drought triggered significant responses in 12 (75%) of the a priori-selected traits. Behavioural traits describing movement (dispersal, locomotion) and diet were sensitive to moderate-intensity drought, as channels fragmented into isolated pools. By comparison, morphological and physiological traits showed little response until surface water was lost, at which point we observed sudden shifts in body size, respiration mode and thermal tolerance. Responses varied widely among TPGs, ranging from population collapses of non-aerial dispersers as channels fragmented to irruptions of small, eurythermic dietary generalists upon extreme dewatering. Our study demonstrates for the first time that relatively small changes in drought intensity can trigger disproportionately large functional shifts in stream communities, suggesting that traits-based approaches could be particularly useful for diagnosing catastrophic ecological responses to global change.

Specialized Information Processing Deficits and Distinct Metabolomic Profiles Following TM-Domain Disruption of Nrg1.

Although there is considerable genetic and pathologic evidence for an association between neuregulin 1 (NRG1) dysregulation and schizophrenia, the underlying molecular and cellular mechanisms remain unclear. Mutant mice containing disruption of the transmembrane (TM) domain of the NRG1 gene constitute a heuristic model for dysregulation of NRG1-ErbB4 signaling in schizophrenia. The present study focused on hitherto uncharacterized information processing phenotypes in this mutant line. Using a mass spectrometry-based metabolomics approach, we also quantified levels of unique metabolites in brain. Across 2 different sites and protocols, Nrg1 mutants demonstrated deficits in prepulse inhibition, a measure of sensorimotor gating, that is, disrupted in schizophrenia; these deficits were partially reversed by acute treatment with second, but not first-, generation antipsychotic drugs. However, Nrg1 mutants did not show a specific deficit in latent inhibition, a measure of selective attention that is also disrupted in schizophrenia. In contrast, in a "what-where-when" object recognition memory task, Nrg1 mutants displayed sex-specific (males only) disruption of "what-when" performance, indicative of impaired temporal aspects of episodic memory. Differential metabolomic profiling revealed that these behavioral phenotypes were accompanied, most prominently, by alterations in lipid metabolism pathways. This study is the first to associate these novel physiological mechanisms, previously independently identified as being abnormal in schizophrenia, with disruption of NRG1 function. These data suggest novel mechanisms by which compromised neuregulin function from birth might lead to schizophrenia-relevant behavioral changes in adulthood.

Potentiation of latent inhibition by haloperidol and clozapine is attenuated in Dopamine D2 receptor (Drd-2)-deficient mice: do antipsychotics influence learning to ignore irrelevant stimuli via both Drd-2 and non-Drd-2 mechanisms?

Whether the dopamine Drd-2 receptor is necessary for the behavioural action of antipsychotic drugs is an important question, as Drd-2 antagonism is responsible for their debilitating motor side effects. Using Drd-2 null mice (Drd2 -/-) it has previously been shown that Drd-2 is not necessary for antipsychotic drugs to reverse D-amphetamine disruption of latent inhibition (LI), a behavioural measure of learning to ignore irrelevant stimuli. Weiner's 'two-headed' model indicates that antipsychotics not only reverse LI disruption, 'disrupted LI', but also potentiate LI when low/absent in controls, 'persistent' LI. We investigated whether antipsychotic drugs haloperidol or clozapine potentiated LI in wild-type controls or Drd2 -/-. Both drugs potentiated LI in wild-type but not in Drd2 -/- mice, suggesting moderation of this effect of antipsychotics in the absence of Drd-2. Haloperidol potentiated LI similarly in both Drd1 -/- and wild-type mice, indicating no such moderation in Drd1 -/-. These data suggest that antipsychotic drugs can have either Drd-2 or non-Drd-2 effects on learning to ignore irrelevant stimuli, depending on how the abnormality is produced. Identification of the non-Drd-2 mechanism may help to identify novel non-Drd2 based therapeutic strategies for psychosis.

N-substituted benztropine analogs: selective dopamine transporter ligands with a fast onset of action and minimal cocaine-like behavioral effects.

Previous studies suggested that differences between the behavioral effects of cocaine and analogs of benztropine were related to the relatively slow onset of action of the latter compounds. Several N-substituted benztropine analogs with a relatively fast onset of effects were studied to assess whether a fast onset of effects would render the effects more similar to those of cocaine. Only one of the compounds increased locomotor activity, and the increases were modest compared with those of 10 to 20 mg/kg cocaine. In rats trained to discriminate 10 mg/kg cocaine from saline none of the compounds produced more than 40% cocaine-like responds up to 2 h after injection. None of the compounds produced place-conditioning when examined up to 90 min after injection, indicating minimal abuse liability. The compounds had 5.6 to 30 nM affinities at the dopamine transporter (DAT), with uniformly lower affinities at norepinephrine and serotonin transporters (from 490-4600 and 1420-7350 nM, respectively). Affinities at muscarinic M(1) receptors were from 100- to 300-fold lower than DAT affinities, suggesting minimal contribution of those sites to the behavioral effects of the compounds. Affinities at histaminic H(1) sites were from 11- to 43-fold lower than those for the DAT. The compounds also had affinity for sigma, 5-hydroxytryptamine(1) (5-HT(1)), and 5-HT(2) receptors that may have contributed to their behavioral effects. Together, the results indicate that a slow onset of action is not a necessary condition for reduced cocaine-like effects of atypical DAT ligands and suggest several mechanisms that may contribute to the reduced cocaine-like efficacy of these compounds.

Mesenchymal stem cells can participate in ischemic neovascularization.

BACKGROUND: Cells from the bone marrow contribute to ischemic neovascularization, but the identity of these cells remains unclear. The authors identify mesenchymal stem cells as a bone marrow-derived progenitor population that is able to engraft into peripheral tissue in response to ischemia. METHODS: A murine model of skin ischemia was used. Bone marrow, blood, and skin were harvested at different time points and subjected to flow cytometric analysis for mesenchymal and hematopoietic markers (n = 3 to 7 per time point). Using a parabiotic model pairing donor green fluorescent protein (GFP)-positive with recipient wild-type mice, progenitor cell engraftment was examined in ischemic tissue by fluorescence microscopy, and engrafted cells were analyzed by flow cytometry for endothelial and mesenchymal markers. In vitro, the ability of both bone marrow- and adipose-derived mesenchymal stem cells to adopt endothelial characteristics was examined by analyzing (1) the ability of mesenchymal stem cells to take up DiI-acetylated low-density lipoprotein and Alexa Fluor lectin, and (2) phenotypic changes of mesenchymal stem cells co-cultured with GFP-labeled endothelial cells or under hypoxic/vascular endothelial growth factor stimulation. RESULTS: In vivo, the bone marrow mesenchymal stem cell population decreased significantly immediately after surgery, with subsequent engraftment of these cells in ischemic tissue. Engrafted cells lacked the panhematopoietic antigen CD45, consistent with a mesenchymal origin. In vitro, bone marrow- and adipose-derived mesenchymal stem cells took up DiI-acetylated low-density lipoprotein and Alexa Fluor lectin, and expressed endothelial markers under hypoxic conditions. CONCLUSIONS: The authors' data suggest that mesenchymal precursor cells can give rise to endothelial progenitors. Consequently, cell-based therapies augmenting the mesenchymal stem cell population could represent powerful alternatives to current therapies for ischemic vascular disease.

Effects of minor laboratory procedures, adrenalectomy, social defeat or acute alcohol on regional brain concentrations of corticosterone.

Concentrations of corticosterone in brain areas of TO strain mice were measured by radioimmunoassay. The studies examined the effects of routine laboratory maneuvers, variation during the circadian peak, adrenalectomy, social defeat and acute injections of alcohol on these concentrations. Brief handling of mice increased corticosterone levels in plasma but not in striatum and reduced those in the hippocampus. Single injections of isotonic saline raised the plasma concentrations to a similar extent as the handling, but markedly elevated concentrations in the three brain regions. Five minutes exposure to a novel environment increased hippocampal and cerebral cortical corticosterone levels and striatal concentrations showed a larger rise. However, by 30 min in the novel environment, plasma concentrations rose further while those in striatum and cerebral cortex fell to control levels and hippocampal corticosterone remained elevated. Over the period of the circadian peak the hippocampal and striatal concentrations paralleled the plasma concentrations but cerebral cortical concentrations showed only small changes. Adrenalectomy reduced plasma corticosterone concentrations to below detectable levels after 48 h but corticosterone levels were only partially reduced in the hippocampus and striatum and remained unchanged in the cerebral cortex. Single or repeated social defeat increased both brain and plasma concentrations after 1 h. Acute injections of alcohol raised the regional brain levels in parallel with plasma concentrations. The results show that measurements of plasma concentrations do not necessarily reflect the levels in brain. The data also demonstrate that corticosterone levels can change differentially in specific brain regions. These results, and the residual hormone seen in the brain after adrenalectomy, are suggestive evidence for a local origin of central corticosterone.

Diabetes increases p53-mediated apoptosis following ischemia.

BACKGROUND: Diabetes impairs the ability of tissue to respond adequately to ischemia. The underlying mechanisms contributing to this impaired response remain unknown. Because increases in apoptosis have been linked to a spectrum of diabetic complications, the authors examined whether programmed cell death is involved in the pathogenesis of poor diabetic tissue responses to ischemia. METHODS: Analysis for apoptosis and levels of proaptotic protein, p53, were performed on streptozocin-induced diabetic mice and wild-type controls in a murine model of soft-tissue ischemia (n = 6). In vitro, chronic hyperglycemic culture conditions were used to test inducibility and reversibility of the diabetic phenotype. Small interfering RNA was used to assess the role of p53. RESULTS: Ischemia-induced apoptosis and p53 levels were increased significantly in diabetic dermal fibroblasts both in vivo and in vitro. Chronic hyperglycemic culture was sufficient to induce the increased apoptotic phenotype, and this was not reversible with long-term normoglycemic conditions. Blocking p53 with small interfering RNA resulted in significant protection against ischemic apoptosis. CONCLUSIONS: These findings suggest that diabetes causes an increased apoptotic response to ischemia through a p53-mediated mechanism. This increase is not reversible by exposure to low-glucose conditions. This suggests that glycemic control alone will be unable to prevent tissue necrosis in diabetic patients and suggests novel therapeutic strategies for this condition.

Decreasing intracellular superoxide corrects defective ischemia-induced new vessel formation in diabetic mice.

Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1alpha by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1alpha modification by methylglyoxal reduced heterodimer formation and HIF1alpha binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes.

Pulsed electromagnetic fields accelerate normal and diabetic wound healing by increasing endogenous FGF-2 release.

BACKGROUND: Chronic wounds, particularly in diabetics, result in significant morbidity and mortality and have a profound economic impact. The authors demonstrate that pulsed electromagnetic fields significantly improve both diabetic and normal wound healing in 66 mice through up-regulation of fibroblast growth factor (FGF)-2 and are able to prevent tissue necrosis in diabetic tissue after an ischemic insult. METHODS: Db/db and C57BL6 mice were wounded and exposed to pulsed electromagnetic fields. Gross closure, cell proliferation, and vascularity were assessed. Cultured medium from human umbilical vein endothelial cells exposed to pulsed electromagnetic fields was analyzed for FGF-2 and applied topically to wounds. Skin flaps were created on streptozocin-induced diabetic mice and exposed to pulsed electromagnetic fields. Percentage necrosis, oxygen tension, and vascularity were determined. RESULTS: Pulsed electromagnetic fields accelerated wound closure in diabetic and normal mice. Cell proliferation and CD31 density were significantly increased in pulsed electromagnetic field-treated groups. Cultured medium from human umbilical vein endothelial cells in pulsed electromagnetic fields exhibited a three-fold increase in FGF-2, which facilitated healing when applied to wounds. Skin on diabetic mice exposed to pulsed electromagnetic fields did not exhibit tissue necrosis and demonstrated oxygen tensions and vascularity comparable to those in normal animals. CONCLUSIONS: This study demonstrates that pulsed electromagnetic fields are able to accelerate wound healing under diabetic and normal conditions by up-regulation of FGF-2-mediated angiogenesis. They also prevented tissue necrosis in response to a standardized ischemic insult, suggesting that noninvasive angiogenic stimulation by pulsed electromagnetic fields may be useful to prevent ulcer formation, necrosis, and amputation in diabetic patients.

Diabetes impairs endothelial progenitor cell-mediated blood vessel formation in response to hypoxia.

BACKGROUND: Diabetics suffer from vascular dysfunction with increased risks of coronary artery disease and peripheral vascular disease secondary to an impaired ability to respond to tissue ischemia. Because endothelial progenitor cells are known to home to sites of ischemia and participate in new blood vessel growth, the authors examined the effects of diabetes on human endothelial progenitor cell function and peripheral tissue signaling in hypoxia, and determined whether these cells might be a useful cell-based therapy for diabetic vascular complications. METHODS: Circulating human endothelial progenitor cells from type 2 diabetic patients and controls were isolated and subjected to in vitro adhesion, migration, and proliferation assays (n = 5). Cell mobilization and recruitment were studied in vivo in diabetic and nondiabetic environments (n = 6). Exogenous human diabetic and normal cells were analyzed for therapeutic efficacy in a murine ischemia model (n = 6). RESULTS: Adhesion, migration, and proliferation of human diabetic endothelial progenitor cells in response to hypoxia was significantly reduced compared with controls. In diabetic mice, cell mobilization from the bone marrow and recruitment into ischemic tissue was significantly reduced compared with controls. Normal cells injected systemically as replacement therapy in a diabetic mouse increased but did not normalize ischemic tissue survival. CONCLUSIONS: These findings suggest that diabetes causes defects in both the endothelial progenitor cell and peripheral tissue responses to hypoxia. These changes in endothelial progenitor cell function and signaling offer a novel explanation for the poor clinical outcome of type 2 diabetics following ischemic events. Based on these findings, it is unlikely that endothelial progenitor cell-based cellular therapies will be able to prevent diabetic complications.

Progress and potential for regenerative medicine.

Regenerative medicine focuses on new therapies to replace or restore lost, damaged, or aging cells in the human body to restore function. This goal is being realized by collaborative efforts in nonmammalian and human development, stem cell biology, genetics, materials science, bioengineering, and tissue engineering. At present, understanding existing reparative processes in humans and exploring the latent ability to regenerate tissue remains the focus in this field. This review covers recent work in limb regeneration, fetal wound healing, stem cell biology, somatic nuclear transfer, and tissue engineering as a foundation for developing new clinical therapies to augment and stimulate human regeneration.

Overview of the role for calreticulin in the enhancement of wound healing through multiple biological effects.

Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRTare substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production.

Skin graft vascularization involves precisely regulated regression and replacement of endothelial cells through both angiogenesis and vasculogenesis.

BACKGROUND: Long-term survival of a skin graft is dependent on eventual revascularization. The authors' aim in the present study was to determine whether skin graft vascularization occurs by (1) simple reconnection of vessels, (2) ingrowth of recipient vasculature, (3) outgrowth of donor-derived vessels, and/or (4) recruitment of bone marrow-derived endothelial progenitor cells. METHODS: Full-thickness skin grafts (1 x 1 cm) were transferred between wild-type FVB/N mice (n = 20) and transgenic tie2/lacZ mice (n = 20), where lacZ expression is controlled by the endothelial specific tie2 promoter, allowing differentiation of recipient and donor endothelial cells. The contribution of endothelial progenitor cells to skin graft neovascularization was determined using a bone marrow transplant model where tie2/lacZ bone marrow was transplanted into wild-type mice (n = 20). RESULTS: Vascular regression in the graft was observed at the periphery starting on day 3 and moving centrally through day 21, sparing graft vessels in the absolute center of the graft. At the same time, vascular ingrowth occurred from the wound bed to replace the regressing vessels. Furthermore, bone marrow-derived endothelial progenitor cells contributed to these new vessels starting as early as day 7. Surprisingly, the contribution of bone marrow-derived vessels to the overall process was approximately 15 to 20 percent of new endothelial cells. CONCLUSIONS: Replacement of the donor graft vasculature by endothelial and endothelial progenitor cells from the recipient along preexisting channels is the predominant mechanism for skin graft revascularization. This mechanism is likely similar for all nonvascularized free grafts and suggests novel strategies for optimizing the vascularization of tissue constructs engineered in vitro.

Hyperglycemia-induced reactive oxygen species and impaired endothelial progenitor cell function.

Vascular complications in diabetes are a significant source of human morbidity and mortality, affecting multiple organ systems and persisting despite tight glucose control. Many of these complications can be linked to impairments in vasculogenesis, the process by which circulating and bone marrow-derived endothelial progenitor cells (EPCs) contribute to new vessel formation. Recent evidence suggests that hyperglycemia alone, through the mitochondrial overproduction of reactive oxygen species (ROS), can induce changes in gene expression and cellular behavior in diabetes. In this review, we examine how hyperglycemia-induced overproduction of ROS could explain EPC impairments observed in diabetes. Experimentally, impairments in EPC function prevent new blood vessel growth and are potentially reversible by manipulations to decrease ROS. Novel strategies aimed at reducing hyperglycemia-induced ROS may be a useful adjuvant to antihyperglycemic therapies in the restoration of vasculogenesis and the prevention of diabetic complications.

The hypothalamopituitary-adrenal axis and alcohol preference.

Effects of alterations in stress hormones and their actions were investigated on alcohol preference, by intraperitoneal administration of RU38486 (a Type II glucocorticoid receptor antagonist, also given by the intracerebroventricular route), spironolactone (a Type I glucocorticoid receptor antagonist), metyrapone (a corticosterone synthesis inhibitor), corticosterone, adrenocorticotropin (ACTH1-39), or intracerebroventricular injection of corticotropin releasing factor (CRF) or a CRF antagonist (alpha-helical CRF9-41). Intracerebroventricular or intraperitoneal administration of RU38486 did not alter the alcohol consumption of mice with high preference for alcohol, or, on first administration, the intake of those with low alcohol preference. When given by repeated intraperitoneal injection however this drug prevented the increase in alcohol consumption seen in "low preference" mice after 3 weeks vehicle injections. Spironolactone did not alter alcohol preference when given by intracerebroventricular or intraperitoneal routes. Repeated, but not single, administration of metyrapone reduced alcohol preference in both high and low preference animals and prevented the increase from low alcohol preference caused by repeated vehicle injections. ACTH1-39 or corticosterone administered by single or repeated intraperitoneal injection, or CRF given i.c.v., did not alter alcohol preference, but the CRF antagonist, alpha-helical CRF9-41, caused a transient increase from low alcohol preference. Blood corticosterone concentrations prior to preference measurements did not correlate with the alcohol preference of the mice. The results indicate that delayed consequences of corticosterone acting on Type II glucocorticoid receptors may be involved in the increases in alcohol preference after injection stress. They also suggest that central actions of CRF may influence the low alcohol consumption of the low alcohol-preferring mice.

Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow-derived cells.

Ischemia is a known stimulus for vascular growth. Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are believed to contribute to new blood vessel growth, but the mechanism for this contribution is unknown. To elucidate how BM cells are able to form new blood vessels, a novel murine model of soft tissue ischemia was developed in lethally irradiated mice with BM reconstituted from either tie2/lacZ or ROSA/green fluorescent protein (GFP) mice (n = 24). BM-derived EPCs were recruited to ischemic tissue within 72 hours, and the extent of recruitment was directly proportional to the degree of tissue ischemia. At 7 days, there were persistently elevated levels of vascular endothelial growth factor (VEGF) (2.5-fold) and circulating VEGF receptor-2/CD11(-) (flk-1(+)/CD11(-)) cells (18-fold) which correlated with increased numbers of BM-derived EPCs within ischemic tissue. The cells were initially located extravascularly as proliferative clusters. By day 14, these clusters coalesced into vascular cords, which became functional vessels by day 21. In vitro examination of human EPCs from healthy volunteers (n = 10) confirmed that EPC proliferation, adhesion, and chemotaxis were all significantly stimulated in hypoxic conditions. We conclude that BM-derived cells produce new blood vessels via localized recruitment, proliferation, and differentiation of circulating cells in a sequence of events markedly different from existing paradigms of angiogenesis.

Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.

The trafficking of circulating stem and progenitor cells to areas of tissue damage is poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) mediates homing of stem cells to bone marrow by binding to CXCR4 on circulating cells. SDF-1 and CXCR4 are expressed in complementary patterns during embryonic organogenesis and guide primordial stem cells to sites of rapid vascular expansion. However, the regulation of SDF-1 and its physiological role in peripheral tissue repair remain incompletely understood. Here we show that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension. HIF-1-induced SDF-1 expression increases the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Blockade of SDF-1 in ischemic tissue or CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury. Discrete regions of hypoxia in the bone marrow compartment also show increased SDF-1 expression and progenitor cell tropism. These data show that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1-induced expression of SDF-1.

Electromagnetic fields increase in vitro and in vivo angiogenesis through endothelial release of FGF-2.

Pulsed electromagnetic fields (PEMF) have been shown to be clinically beneficial, but their mechanism of action remains unclear. The present study examined the impact of PEMF on angiogenesis, a process critical for successful healing of various tissues. PEMF increased the degree of endothelial cell tubulization (sevenfold) and proliferation (threefold) in vitro. Media from PEMF cultures had a similar stimulatory effect, but heat denaturation ablated this activity. In addition, conditioned media was able to induce proliferative and chemotactic changes in both human umbilical vein endothelial cells and fibroblasts, but had no effect on osteoblasts. Angiogenic protein screening demonstrated a fivefold increase in fibroblast growth factor beta-2 (FGF-2), as well as smaller increases in other angiogenic growth factors (angiopoietin-2, thrombopoietin, and epidermal growth factor). Northern blot analysis demonstrated an increase in FGF-2 transcription, and FGF-2 neutralizing antibody inhibited the effects of PEMF. In vivo, PEMF exposure increased angiogenesis more than twofold. We conclude that PEMF augments angiogenesis primarily by stimulating endothelial release of FGF-2, inducing paracrine and autocrine changes in the surrounding tissue. These findings suggest a potential role for PEMF in therapeutic angiogenesis.