Jamie P. Levine

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.

Cellular Dysfunction in the Diabetic Fibroblast: Impairment in Migration, Vascular Endothelial Growth Factor Production, and Response to Hypoxia

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.

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 β‐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.

Studies in cranial suture biology: regional dura mater determines overlying suture biology.

&NA; The influence of dura mater on adjacent cranial sutures is significant. By better understanding the mechanisms of normal suture fusion and the role of the dura mater, it may be possible to delineate the events responsible for the premature suture fusion seen in craniosynostosis. In the Sprague‐Dawley rat, the posterior frontal suture normally fuses between 12 and 20 days of postnatal life and has proved to be an excellent model to describe normal suture fusion. The purpose of this study was to document the critical role that the dura mater‐suture complex may play on cranial suture biology. Forty Sprague‐Dawley rats at 8 days of age were divided into two groups of 20 animals each. The control group (group A) had surgical disruption of the dura matercalvarial interface. This was accomplished by elevating a strip of cranium inclusive of the posterior frontal and sagittal sutures and replacement of the cranial strip back to its anatomic position, all with the dura mater left intact. The experimental group (group B) had the same calvarial elevation (strip craniectomy), but the sutural anatomy/alignment was rotated 180 degrees. This rotation placed the posterior frontal suture into the sagittal sutures anatomic position and the sagittal suture into the posterior frontal sutures anatomic position. All of these procedures were accomplished by leaving the underlying dura mater intact. Animals were killed at 20, 30, 40, and 50 days (12, 22, 32, and 42 days postoperatively), and tissue sections were examined with hematoxylin and eosin staining. Group A (control) showed normal but delayed suture activity. The posterior frontal suture fused, and the sagittal suture remained patent. Fusion was delayed, not beginning before 20 days (12 days postoperative) and showing complete fusion between 30 and 40 days. Group B (180degree calvarial rotation) demonstrated that the suture in the posterior frontal anatomic position (actual sagittal suture) fused between 20 and 40 days, whereas the suture in the sagittal anatomic position (actual posterior‐frontal suture) remained patent throughout the study. This study demonstrates that the location of the dura mater‐suture complex is important in determining either suture patency or closure in this model. Normal closure of the suture overlying the posterior frontal dura mater demonstrates that the dura mater itself, or forces derived in specific cranial locations, determines the overlying suture biology. (Plast. Reconstr. Surg. 101: 1441, 1998.)

Studies in Cranial Suture Biology: Part I. Increased Immunoreactivity for TGF-β Isoforms (β1, β2, and β3) During Rat Cranial Suture Fusion†

The mechanisms involved in normal cranial suture development and fusion as well as the pathophysiology of craniosynostosis, a premature fusion of the cranial sutures, are not well understood. Transforming growth factor‐β isoforms (TGF‐β1, β2, and β3) are abundant in bone and stimulate calvarial bone formation when injected locally in vivo. To gain insight into the role of these factors in normal growth and development of cranial sutures and the possible etiology of premature cranial suture fusion, we examined the temporal and spatial expression of TGF‐β isoforms during normal cranial suture development in the rat. In the Sprague‐Dawley rat, only the posterior frontal cranial suture undergoes fusion between 12 and 22 days of age, while all other cranial sutures remain patent. Therefore, immunohistochemical analysis of the fusing posterior frontal suture was compared with the patent sagittal suture at multiple time points from the fetus through adult. Whereas the intensity of immunostaining was the same in the posterior frontal and sagittal sutures in the fetal rat, there was increased immunoreactivity for TGF‐β isoforms in the actively fusing posterior frontal suture compared with the patent sagittal suture starting 2 days after birth and continuing until approximately 20 days. There were intensely immunoreactive osteoblasts present during fusion of the posterior frontal suture. In contrast, the patent sagittal suture was only slightly immunoreactive. A differential immunostaining pattern was observed among the TGF‐β isoforms; TGF‐β2 was the most immunoreactive isoform and was also most strongly associated with osteoblasts adjacent to the dura and the margin of the fusing suture. Since the increased expression of TGF‐β2 during suture fusion suggested a possible regulatory role, recombinant TGF‐β2 was added directly to the posterior frontal and sagittal sutures in vivo to determine if suture fusion could be initiated. Exogenously added TGF‐β2 stimulated fusion of the ectocranial surface of the posterior frontal suture. These data provide evidence for a regulatory role for these growth factors in cranial suture development and fusion. Additionally, the intense immunostaining for TGF‐β2 in the dura mater underlying the fusing suture supports a role for the dura mater in suture fusion. It is possible that premature or excessive expression of these factors may be involved in the etiopathogenesis of craniosynostosis and that modulation of the growth factor profile at the suture site may have potential therapeutic value.

Studies in cranial suture biology : up-regulation of transforming growth factor-beta1 and basic fibroblast growth factor mRNA correlates with posterior frontal cranial suture fusion in the rat

&NA; The mechanisms involved in normal cranial suture development and fusion as well as in the pathophysiology of craniosyostosis are not well understood. The purpose of this study was to investigate the expression of several cytokines—transforming growth factor‐beta‐1 (TGF‐&bgr;1), basic fibroblast growth factor (bFGF), and interleukin‐6 (IL‐6)—during cranial suture fusion. TGF‐&bgr; exists in three mammalian isoforms that are abundant in bone and stimulate calvarial bone formation when delivered locally. Other bone growth factors including basic fibroblast growth factor and the interleukins regulate bone growth and are mitogenic for bone marrow cells and osteoblasts. The involvement of growth factors in the pathophysiology of craniosynostosis is supported by recent genetics data linking fibroblast growth factor receptor mutations to syndromal craniosynostoses. In this experimental study, in situ hybridization was used to localize and quantify the gene expression of TGF‐&bgr;1, bFGF, and IL‐6 during cranial suture fusion. In the Sprague‐Dawley rat, the posterior frontal cranial suture normally undergoes fusion between 12 and 22 days of age, whereas all other cranial sutures remain patent. All in situ analyses of fusing posterior frontal sutures were compared with the patent, control, sagittal sutures. Posterior frontal and sagittal sutures, together with underlying dura, were harvested from rats at 8, 12, 16, and 35 days of postnatal life to analyze posterior frontal suture activity before, during, and after fusion. In situ hybridization was performed on frozen sections of these specimens using DNA probes specific for TGF‐&bgr;1, bFGF, and IL‐6 mRNA. A negative control probe to IL‐6 in the sense orientation was also used to validate the procedure. Cells expressing cytokine‐specific mRNA were quantified (in cells positive per 10‐1 mm2) and analyzed using the unpaired Students t test. Areas encompassing the fibrous suture and the surrounding bone plates were analyzed for cellular mRNA activity. IL‐6 mRNA expression showed a minimal rise in the posterior frontal suture at days 12 and 16, with an average count of 10 and 6 cells per 10‐1 mm2, respectively. The sagittal suture remained negative for IL‐6 mRNA at all time points. TGF‐&bgr;1 and bFGF analyses were most interesting, showing marked increases specifically in the posterior frontal suture during the time of active suture fusion. On postnatal day 8, a 1.5‐fold increase in posterior frontal suture TGF‐&bgr;1 mRNA was found compared with sagittal sutures (p = 0.1890, unpaired Students t test). This difference was increased 26‐fold on day 12 in posterior frontal suture TGF‐&bgr;1 expression (p = 0.0005). By day 35, posterior frontal suture TGF‐&bgr;1 mRNA had nearly returned to prefusion levels, whereas TGF‐&bgr;1 mRNA levels in the sagittal suture remained low. A similar upregulation of bFGF mRNA, peaking at day 12, was observed in posterior frontal but not sagittal sutures (p = 0.0003). Furthermore, both TGF‐&bgr;1 and bFGF mRNA samples with intact dura showed an intense dural mRNA expression in the time preceding and during active posterior frontal suture fusion but not in sagittal tissues. Our data demonstrate that TGF‐&bgr;1 and bFGF mRNA are up‐regulated in cranial suture fusion, possibly signaling in a paracrine fashion from dura to suture. TGF‐&bgr;1 and bFGF gene expression were dramatically increased both in and surrounding the actively fusing suture and followed the direction of fusion from endocranial to epicranial. These experimental data on bone growth factors support the recent human genetics data linking growth factor/fibroblast growth factor receptor deletions to syndromal craniosynostoses. The ultimate aim of these studies is to understand the underlying mechanisms regulating suture growth, development, and fusion so surgeons may one day manipulate the biology of premature cranial suture fusion. (Plast. Reconstr. Surg. 101: 1431, 1998.)

The use of acellular dermal matrix in immediate two-stage tissue expander breast reconstruction.

Background: Acellular dermal matrix is commonly used in implant-based breast reconstruction to allow for quicker tissue expansion with better coverage and definition of the lower pole of the breast. This study was performed to analyze complications associated with its use in immediate two-stage, implant-based breast reconstruction and to subsequently develop guidelines for its use. Methods: A retrospective analysis of 628 consecutive immediate two-stage tissue expander breast reconstructions at a single institution over a 3-year period was conducted. The reconstructions were divided into two groups: reconstruction with acellular dermal matrix and reconstruction without it. Demographic information, patient characteristics, surface area of acellular dermal matrix, and complications were analyzed and compared. Results: A total of 407 patients underwent 628 immediate two-stage, implant-based breast reconstructions; 442 reconstructions (70.3 percent) used acellular dermal matrix and 186 (29.6 percent) did not. The groups had similar patient characteristics; however, major complications were significantly increased in the acellular dermal matrix group (15.3 versus 5.4 percent; p = 0.001). These complications included infection requiring intravenous antibiotics (8.6 versus 2.7 percent; p = 0.001), flap necrosis requiring excision (6.7 versus 2.7 percent; p = 0.015), and explantation of the tissue expander (7.7 versus 2.7 percent; p = 0.004). Conclusions: Use of acellular dermal matrix in immediate two-stage, implant-based breast cancer reconstruction is associated with a significant increase in major complications. Therefore, it should only be used in specific patients and in minimal amounts. Indications for its use include single-stage permanent implant reconstruction and inadequate local muscle coverage of the tissue expander. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Studies in cranial suture biology: part II. Role of the dura in cranial suture fusion.

&NA; The biology underlying normal and premature cranial suture fusion remains unknown. The purpose of this study was to investigate the role of the dura mater in cranial suture fusion. In the Sprague Dawley rat model, the posterior frontal cranial suture fuses between 10 and 20 days of postnatal life. The effect of separating the posterior frontal cranial suture from its underlying dura mater with an intervening silastic sheet was studied. Sixty rat pups, age 8 days, were divided into four groups of 15. Group A served as unoperated controls. Group B, the experimental group, underwent craniotomy, dural elevation, and insertion of a silicone sheet between the posterior frontal cranial suture and the underlying dura. Two operative sham groups were included. Group C underwent craniotomy and dural deflection only. Group D underwent craniotomy alone without dural deflection. The rats were sacrificed at 15, 22, and 30 days of age. The results showed that the unoperated animals (group A) demonstrated normal initiation of suture fusion at 15 days and complete fusion by 22 days. Group B animals, with silicone sheet barriers placed, showed persistent patency of sutures at 22 days. Initiation of suture fusion was delayed until 30 days. Sham group C, animals with craniotomy and dural deflection, showed that initiation of fusion was delayed until 22 days with complete fusion by 30 days of age. Sham group D, craniotomy alone, had the same normal temporal sequence of suture fusion as the unoperated control group A. These data indicate that normal cranial suture fusion is delayed when the suture—dural interaction is interrupted by a surgically placed barrier or by simple dural deflection. Furthermore, interaction between the dura and the overlying suture appears to direct suture fusion. (Plast. Reconstr. Surg. 97: 693, 1996.)

Auricular reconstruction: indications for autogenous and prosthetic techniques.

LEARNING OBJECTIVES After studying this article, the participant should be able to: 1. Describe the alternatives for auricular reconstruction. 2. Discuss the pros and cons of autogenous reconstruction of total or subtotal auricular defects. 3. Enumerate the indications for prosthetic reconstruction of total or subtotal auricular defects. 4. Understand the complexity of and the expertise required for prosthetic reconstruction of auricular defects. The indications for autogenous auricular reconstruction versus prosthetic reconstruction with osseointegrated implant-retained prostheses were outlined in Plastic and Reconstructive Surgery in 1994 by Wilkes et al. of Canada, but because of the relatively recent Food and Drug Administration approval (1995) of extraoral osseointegrated implants, these indications had not been examined by a surgical unit in the United States. The purpose of this article is to present an evolving algorithm based on an experience with 98 patients who underwent auricular reconstruction over a 10-year period. From this experience, the authors conclude that autogenous reconstruction is the procedure of choice in the majority of pediatric patients with microtia. Prosthetic reconstruction of the auricle is considered in such pediatric patients with congenital deformities for the following three relative indications: (1) failed autogenous reconstruction, (2) severe soft-tissue/skeletal hypoplasia, and/or (3) a low or unfavorable hairline. A fourth, and in our opinion the ideal, indication for prosthetic ear reconstruction is the acquired total or subtotal auricular defect, most often traumatic or ablative in origin, which is usually encountered in adults. Although prosthetic reconstruction requires surgical techniques that are less demanding than autogenous reconstruction, construction of the prosthesis is a time-consuming task requiring experience and expertise. Although autogenous reconstruction presents a technical challenge to the surgeon, it is the prosthetic reconstruction that requires lifelong attention and may be associated with late complications. This article reports the first American series of auricular reconstruction containing both autogenous and prosthetic methods by a single surgical team.