Jennifer M. Capla

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.

Human Endothelial Progenitor Cells From Type II Diabetics Exhibit Impaired Proliferation, Adhesion, and Incorporation Into Vascular Structures

Background—The recent discovery of circulating endothelial progenitor cells (EPCs) has altered our understanding of new blood vessel growth such as occurs during collateral formation. Because diabetic complications occur in conditions in which EPC contributions have been demonstrated, EPC dysfunction may be important in their pathophysiology. Methods and Results—EPCs were isolated from human type II diabetics (n=20) and age-matched control subjects (n=20). Proliferation of diabetic EPCs relative to control subjects was decreased by 48% (P <0.01) and inversely correlated with patient levels of hemoglobin A1C (P <0.05). Diabetic EPCs had normal adhesion to fibronectin, collagen, and quiescent endothelial cells but a decreased adherence to human umbilical vein endothelial cells activated by tumor necrosis factor-&agr; (TNF-&agr;) (P <0.05). In a Matrigel assay, diabetic EPCs were 2.5 times less likely to participate in tubule formation compared with controls (P <0.05). Conclusions—These findings suggest that type II diabetes may alter EPC biology in processes critical for new blood vessel growth and may identify a population at high risk for morbidity and mortality after vascular occlusive events.

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.

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 × 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.

Selective recruitment of endothelial progenitor cells to ischemic tissues with increased neovascularization.

Tissue ischemia remains a common problem in plastic surgery and one for which proangiogenic approaches have been investigated. Given the recent discovery of circulating endothelial stem or progenitor cells that are able to form new blood vessels, the authors sought to determine whether these cells might selectively traffic to regions of tissue ischemia and induce neovascularization. Endothelial progenitor cells were isolated from the peripheral blood of healthy human volunteers and expanded ex vivo for 7 days. Elevation of a cranially based random-pattern skin flap was performed in nude mice, after which they were injected with fluorescent-labeled endothelial progenitor cells (5 × 105; n = 15), fluorescent-labeled human microvascular endothelial cells (5 × 105; n = 15), or media alone (n = 15). Histologic examination demonstrated that endothelial progenitor cells were recruited to ischemic tissue and first appeared by postoperative day 3. Subsequently, endothelial progenitor cell numbers increased exponentially over time for the remainder of the study [0 cells/mm2 at day 0 (n = 3), 9.6 ± 0.9 cells/mm2 at day 3 (n = 3), 24.6 ± 1.5 cells/mm2 at day 7 (n = 3), and 196.3 ± 9.6 cells/mm2 at day 14 (n = 9)]. At all time points, endothelial progenitor cells localized preferentially to ischemic tissue and healing wound edges, and were not observed in normal, uninjured tissues. Endothelial progenitor cell transplantation led to a statistically significant increase in vascular density in ischemic tissues by postoperative day 14 [28.7 ± 1.2 in the endothelial progenitor cell group (n = 9) versus 18 ± 1.1 in the control media group (n = 9) and 17.7 ± 1.0 in the human microvascular endothelial cell group (n = 9; p < 0.01)]. Endothelial progenitor cell transplantation also showed trends toward increased flap survival [171.2 ± 18 mm2 in the endothelial progenitor cell group (n = 12) versus 134.2 ± 10 mm2 in the media group (n = 12) and 145.0 ± 13 mm2 in the human microvascular endothelial cell group (n = 12)], but this did not reach statistical significance. These findings indicate that local tissue is- chemia is a potent stimulus for the recruitment of circulating endothelial progenitor cells. Systemic delivery of endothelial progenitor cells increased neovascularization and suggests that autologous endothelial progenitor cell transplantation may have a role in the salvage of ischemic tissue.

Increased circulating AC133+ CD34+ endothelial progenitor cells in children with hemangioma.

UNLABELLED Hemangioma is the most common soft-tissue tumor of infancy. Despite the frequency of these vascular tumors, the origin of hemangioma-endothelial cells is unknown. Circulating endothelial progenitor cells (EPCs) have recently been identified as vascular stem cells with the capacity to contribute to postnatal vascular development. We have attempted to determine whether circulating EPCs are increased in hemangioma patients and thereby provide insight into the role of EPCs in hemangioma growth. METHODS AND RESULTS Peripheral blood mononuclear cells (PBMCs) were isolated from hemangioma patients undergoing surgical resection (N = 5) and from age-matched controls (N = 5) undergoing strabismus correction surgery. PBMCs were stained with fluorescent-labeled antibodies for AC133, CD34, and VEGFR2/KDR. Fluorescent-labeled isotype antibodies served as negative controls. Histologic sections of surgical specimens were stained with the specific hemangioma markers Glut1, CD32, and merosin, to confirm the diagnosis of common hemangioma of infancy. EPCs harvested from healthy adult volunteers were stained with Glut1, CD32, and merosin, to assess whether cultured EPCs express known hemangioma markers. Hemangioma patients had a 15-fold increase in the number of circulating CD34 AC133 dual-staining cells relative to controls (0.78+/-0.14% vs.0.052+/-0.017%, respectively). Similarly, the number of PBMCs that stained positively for both CD34 and KDR was also increased in hemangioma patients (0.49+/-0.074% vs. 0.19+/-0.041% in controls). Cultured EPCs stained positively for the known hemangioma markers Glut1, CD32, merosin. CONCLUSIONS This is the first study to suggest a role for EPCs in the pathogenesis of hemangioma. Our results imply that increased levels of circulating EPCs may contribute to the formation of this vascular tumor.

Stem cells and distraction osteogenesis: Endothelial progenitor cells home to the ischemic generate in activation and consolidation

Background: Ischemia is a limiting factor during distraction osteogenesis. The authors sought to determine the extent of ischemia in the distraction zone and whether endothelial progenitor cells home to the distraction zone and participate in local vasculogenesis. Methods: Laser Doppler imaging was used to assess the extent of blood flow in the distraction zone in gradually distracted, immediately distracted, and osteotomized rat mandibles during activation and consolidation. Animals (n = 50; 25 rats with unilateral gradual distraction and contralateral osteotomy as an internal control, and 25 rats with unilateral immediate distraction) were examined on postoperative days 4, 6, and 8 of activation, and after 1 and 2 weeks of consolidation. Endothelial progenitor cells isolated from human peripheral blood were labeled with fluorescent DiI dye, and 0.5 × 106 cells were injected intra-arterially under direct vision into each carotid artery at the start of activation in nude rats (n = 18) that then underwent the distraction protocol outlined above. Results: Doppler flow analysis demonstrated relative ischemia during the activation period in the distraction osteogenesis group and increased blood flow in the osteotomized control group as compared with flow in a normal hemimandible [normal, 1 (standardized); distraction osteogenesis, 0.58 ± 0.05; control, 2.58 ± 0.21; p < 0.05 for both results]. We observed a significantly increased endothelial progenitor cell population at the generate site versus controls at midactivation and at 1 and 2 weeks of consolidation [25 ± 1.9 versus 1 ± 0.3 DiI-positive cells per high-power field (p < 0.05), 124 ± 21 versus 8 ± 4 DiI-positive cells per high-power field (p < 0.05), and 106 ± 18 versus 9 ± 3 DiI-positive cells per high-power field (p < 0.05), respectively]. Conclusions: These data suggest that the distraction zone becomes relatively ischemic during activation and that endothelial progenitor cells home to the ischemic generate site during the activation phase and remain during the consolidation phase. Selective expansion of these stem cells may be useful in overcoming ischemic limitations of distraction osteogenesis. Moreover, their homing capability may be used to effect site-specific transgene delivery to the generate.

Discussion: Randomized sham-controlled trial to evaluate the safety and effectiveness of a high-intensity focused ultrasound device for noninvasive body sculpting.

W ill liposuction become obsolete? Fueled by the success of effective minimally invasive and noninvasive treatments for aesthetic management of the face, the quest for effective nonsurgical body contouring technology continues at a robust pace. To date, no noninvasive body contouring treatment has assumed a prominent role in plastic surgery practice. Jewell et al. present data from a randomized controlled trial to demonstrate the efficacy of a high-intensity focused ultrasound device for reducing subcutaneous fat deposits. This study is a thorough investigation of the LipoSonix high-intensity focused ultrasound technology (Medicis Technologies Corp., Scottsdale, Ariz.), demonstrating both safety and efficacy in a blinded prospective trial. The authors are to be commended for collecting well-controlled data to test the effect of this device. According to the 2010 report of plastic surgery statistics from the American Society of Plastic Surgeons, the number of patients seeking cosmetic minimally invasive procedures has increased from 5,500,446 in 2000 to 11,561,449 in 2010.1 This 110 percent increase is indicative of patients seeking the “quick fix” that allows them to improve areas of concern with decreased downtime and minimal risk. At 203,106 cases, liposuction is still one of the top five cosmetic surgical procedures performed in 2010, and while it is touted as “minimally invasive,” many technologies have been explored in an effort to improve body contour and tighten skin with less recovery and risk. In Jewell et al.’s article, we see data from a multicenter, randomized, sham-controlled, single-blinded trial of 180 patients designed to determine the effectiveness of high-intensity focused ultrasound on trunk aesthetics. The primary outcome measure was reduction in waist circumference at 12 weeks. In patients treated at the highest energy level (59 J/cm2) in the “per protocol” group, in which maximal effect was seen, there was a statistically significant decrease in waist circumference of 2.52 cm compared with a decrease in the sham control group of 1.21 cm. One potential source of error in this study is the use of a standard measuring tape to determine waist circumference.2 Other modalities, such as three-dimensional photography or magnetic resonance imaging assessment, may be considered for future studies.3 In particular, magnetic resonance imaging will show precise changes in the thickness of the abdominal subcutaneous tissues after treatment. A major challenge in the evaluation of noninvasive body contouring treatments is establishing standard methods of assessment that can provide accurate and reproducible results. One other variable that may affect the results presented in this article is that the patients in this study population did not respond as well as other patients might have. Indeed, a prior high-intensity focused ultrasound study of 282 patients by Fatemi, one of the current authors, revealed an average circumference reduction in the abdomen and waist of 4 to 5 cm.4 While effective, the change in waist circumference induced by high-intensity focused ultrasound is relatively small compared with that in the sham-treated patients. This leads to two important questions. First, why does the sham group have a waist reduction of 1.21 cm? Second, how does high-intensity focused ultrasound fit into our aesthetic armamentarium? With regard to the changes in the sham group, the authors stated that patients agreed to maintain their current diet and exercise regimens without any changes during the study period. In their Discussion, the authors do suggest that this decrease in waist circumference in control subjects may be

Discussion: Prospective clinical study reveals significant reduction in triglyceride level and white blood cell count after liposuction and abdominoplasty and no change in cholesterol levels.

T concept that liposuction may potentially play a therapeutic role in the treatment of medical comorbidities has intrigued the plastic surgery community and the public. Fat is known to be metabolically active. Reduction of body mass may reduce this activity and improve associated metabolic syndrome. There is no doubt that bariatric surgery has revolutionized the treatment of obesity. In so doing, it has been found that medical comorbidities associated with obesity have either been significantly improved or resolved with bariatric surgery. The rate of remission of type 2 diabetes has reached levels of 76.8 percent and was significantly improved in 86 percent of patients. In addition, hypertension, high cholesterol, and sleep apnea were reduced or eliminated in 78.5, 70, and 85.7 percent, respectively.1 These facts have led to a change in the name of the society after 25 years to the American Society for Metabolic and Bariatric Surgery. Can removal of subcutaneous adipose tissue effect positive metabolic changes? Previous studies have looked at the use of liposuction in the treatment of metabolic derangements associated with obesity. A study published in The New England Journal of Medicine in 2004 concluded that large-volume abdominal liposuction performed in 15 obese women did not significantly impact metabolic abnormalities associated with obesity, cardiovascular risk factors, insulin resistance, or markers of inflammation.2 Reviewers of this study criticized the small patient cohort, inadequate long-term follow-up, and lack of removal of visceral fat as explanations for negative findings.3 Looking at a subset of these patients long term, however, showed no improvement in the metabolic endpoints from baseline over time.4 On the contrary, Giugliano et al. found that large-volume liposuction significantly decreased insulin resistance and concentrations of vascular inflammatory markers such as interleukin-6, interleukin-18, tumor necrosis factor, and C-reactive protein in healthy obese women.5 Interestingly, one group performed “mega-lipoplasty” with aspirate volumes of 10 to 26 liters on morbidly obese women and found that the only parameter with a significant impact was a reduction in insulin resistance.6 Others have studied large-volume liposuction in nonobese subjects and found improvement in the major lipoproteins associated with dyslipidemia in obesity.7 The major weaknesses of the above studies lie in the variability of the subjects. In patients who are nonobese, change may be seen, but it is of no significance if the baseline numbers were within normal limits. In patients who are morbidly obese, it appears that an inadequate amount of fat is removed to significantly impact a change in body mass index and therefore a lack of change in associated comorbidities. In addition, the number of subjects in each group was limited to a small sample size, yielding studies with limited power. The authors of this study present a prospective study of 322 healthy patients to determine whether reduction of subcutaneous adiposity by means of liposuction and/or abdominoplasty has an impact on lipid levels. Fasting blood tests were performed preoperatively and at 1 and 3 months after surgery. Of the 229 patients who underwent liposuction alone, 152 completed the study, for an inclusion rate of 66.4 percent. The mean body mass index of the patients in this study was 26.2