Alex K. Wong

Human Gingiva-Derived Mesenchymal Stem Cells Elicit Polarization of M2 Macrophages and Enhance Cutaneous Wound Healing

Increasing evidence has supported the important role of mesenchymal stem cells (MSCs) in wound healing, however, the underlying mechanism remains unclear. Recently, we have isolated a unique population of MSCs from human gingiva (GMSCs) with similar stem cell‐like properties, immunosuppressive, and anti‐inflammatory functions as human bone marrow‐derived MSCs (BMSCs). We describe here the interplay between GMSCs and macrophages and the potential relevance in skin wound healing. When cocultured with GMSCs, macrophages acquired an anti‐inflammatory M2 phenotype characterized by an increased expression of mannose receptor (MR; CD206) and secretory cytokines interleukin (IL)‐10 and IL‐6, a suppressed production of tumor necrosis factor (TNF)‐α, and decreased ability to induce Th‐17 cell expansion. In vivo, we demonstrated that systemically infused GMSCs could home to the wound site in a tight spatial interaction with host macrophages, promoted them toward M2 polarization, and significantly enhanced wound repair. Mechanistically, GMSC treatment mitigated local inflammation mediated by a suppressed infiltration of inflammatory cells and production of IL‐6 and TNF‐α, and an increased expression of IL‐10. The GMSC‐induced suppression of TNF‐α secretion by macrophages appears to correlate with impaired activation of NFκB p50. These findings provide first evidence that GMSCs are capable to elicit M2 polarization of macrophages, which might contribute to a marked acceleration of wound healing. STEM CELLS 2010;28:1856–1868

A systematic review and meta-analysis of complications associated with acellular dermal matrix-assisted breast reconstruction

Background:Multiple outcome studies have been published on the use of acellular dermal matrix (ADM) in breast reconstruction with disparate results. The purpose of this study was to conduct a systematic review and meta-analysis to determine an aggregate estimate of risks associated with ADM-assisted breast reconstruction. Methods:The MEDLINE, Web of Science, and Cochrane Library databases were queried, and relevant articles published up to September 2010 were analyzed based on specific inclusion criteria. Seven complications were studied including seroma, cellulitis, infection, hematoma, skin flap necrosis, capsular contracture, and reconstructive failure. A pooled random effects estimate for each complication and 95% confidence intervals (CI) were derived. For comparisons of ADM and non-ADM, the pooled random effects odds ratio (OR) and 95% CI were derived. Heterogeneity was measured using the I2 statistic. Results:Sixteen studies met the inclusion criteria. The pooled complication rates were seroma (6.9%; 95% CI, 5.3%–8.8%), cellulitis (2.0%; 95% CI, 1.2%–3.1%), infection (5.7%; 95% CI, 4.3%–7.3%), skin flap necrosis (10.9%; 95% CI, 8.7%–13.5%), hematoma (1.3%; 95% CI, 0.6%–2.4%), capsular contracture (0.6%; 95% CI, 0.1%–1.7%), and reconstructive failure (5.1%; 95% CI, 3.8%–6.7%). Five studies reported findings for both the ADM and non-ADM patients and were used in the meta-analysis to calculate pooled OR. ADM-assisted breast reconstructions had a higher likelihood of seroma (pooled OR, 3.9; 95% CI, 2.4–6.2), infection (pooled OR, 2.7; 95% CI, 1.1–6.4), and reconstructive failure (pooled OR, 3.0; 95% CI, 1.3–6.8) than breast reconstructions without the use of ADM. The relation of ADM use to hematoma (pooled OR, 2.0; 95% CI, 0.8–5.2), cellulitis (pooled OR, 2.0; 95% CI, 0.9–4.3), and skin flap necrosis (pooled OR, 1.9; 95% CI, 0.6–5.4) was inconclusive. Conclusions:In the studies evaluated, ADM-assisted breast reconstructions exhibited a higher likelihood of seroma, infection, and reconstructive failure than prosthetic-based breast reconstructions using traditional musculofascial flaps. ADM is associated with a lower rate of capsular contracture. A careful risk/benefit analysis should be performed when choosing to use ADM in implant-based breast reconstruction.

Histologic Analysis of Angiogenesis and Lymphangiogenesis in Acellular Human Dermis

Background: Acellular dermal matrix (AlloDerm) is used frequently in a variety of reconstructive procedures. Although it is clear that AlloDerm is revascularized by host tissues, the mechanisms by which vascularization and tissue incorporation occur remain essentially unknown. The purpose of this experiment was to delineate the time course and composition of host cell infiltrate into the matrix of an AlloDerm composite flap. Methods: A flap based on the superficial inferior epigastric pedicle in the rat was developed and used to obtain tissue specimens at 3, 7, and 14 days after implantation of AlloDerm. Histology, bromodeoxyuridine incorporation, and immunohistologic assays were used to temporally characterize the appearance of myofibroblasts, endothelial cells, and lymphatic endothelial cells within the matrix. Results: Active host cell proliferation occurs within the matrix at 7 days after implantation. The total number of both host cells and myofibroblasts increased by 8-fold between days 3 and 14. There was a 4-fold increase in endothelial cells between days 3 and 7 but no significant increase at day 14. Putative lymphatic channels were identified within the matrix by 14 days and confirmed using immunohistochemistry for Prox-1, a well-established lymphatic endothelial cell marker. Conclusions: The host response to AlloDerm parallels normal wound healing. Host cell infiltrate increases steadily over a 14-day period. By 7 days after implantation, a large number of CD31+ endothelial cells have infiltrated the matrix and early vessels are abundantly present. These vessels continue to mature by day 14. Finally, the authors show that AlloDerm composite flaps also support infiltration and development of a lymphatic network.

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5-30 times the IC50 (30 micrograms ml-1) of parental cells. The vinblastine-resistant parasites were also resistant to puromycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug efflux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdr1, and demonstrate that this gene is amplified on an extrachromosomal circle of 35-40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr1 and 83% identity with the L. donovani ldmdr1 gene. The lemdr1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.

Excessive tumor-elaborated VEGF and its neutralization define a lethal paraneoplastic syndrome

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and key regulator of both physiologic and pathologic (e.g., tumor) angiogenesis. In the course of studies designed to assess the ability of constitutive VEGF to block tumor regression in an inducible RAS melanoma model, mice implanted with VEGF-expressing tumors sustained high morbidity and mortality that were out of proportion to the tumor burden. Documented elevated serum levels of VEGF were associated with a lethal hepatic syndrome characterized by massive sinusoidal dilation and endothelial cell proliferation and apoptosis. Systemic levels of VEGF correlated with the severity of liver pathology and overall clinical compromise. A striking reversal of VEGF-induced liver pathology and prolonged survival were achieved by surgical excision of VEGF-secreting tumor or by systemic administration of a potent VEGF antagonist (VEGF-TRAPR1R2), thus defining a paraneoplastic syndrome caused by excessive VEGF activity. Moreover, this VEGF-induced syndrome resembles peliosis hepatis, a rare human condition that is encountered in the setting of advanced malignancies, high-dose androgen therapy, and Bartonella henselae infection. Thus, our findings in the mouse have suggested an etiologic role for VEGF in this disease and may lead to diagnostic and therapeutic options for this debilitating condition in humans.

Use of human acellular dermal matrix in implant- based breast reconstruction: evaluating the evidence.

The use of acellular dermal matrix (ADM) in implant based breast reconstruction has become increasingly popular to the point that a subset of surgeons use ADM for virtually every tissue expander/implant based reconstruction. While there may be a number of perceived and anecdotal advantages such as decreased post-operative pain, increased initial expander fill volume, and improved aesthetic outcome, it remains unclear as to whether there is sufficient evidence to support these as well as other claims or its routine use. In this review, we identified all papers in the PubMed and Medline databases that addressed outcomes of the use of ADM in single and multiple staged implant based breast reconstruction. Papers were evaluated for any claim of benefit in using ADM in breast reconstruction. The following perceived advantages were supported solely by anecdotal reports and opinions: reduction in post-op pain, decreased operative time, precise control of the lateral and IMF, maximal use of mastectomy skin flaps, and improved lower pole expansion. There was inconsistent data for commonly perceived advantages, such as: eliminating the need for expanders, increased initial fill volumes, fewer expansions, faster time to reconstruction completion, decreased rate of revision, and improved aesthetic outcome. We found consistent support for a decreased incidence of capsular contracture; however the existing reports have limited long term follow-up. Despite the many heralded benefits of ADM in breast reconstruction, the data supporting these claims is mostly anecdotal. Both long term outcomes and randomized controlled prospective studies are needed in order to definitively evaluate the perceived advantages of ADM in breast reconstruction.

ANALYSIS OF RISK FACTORS ASSOCIATED WITH MICROVASCULAR FREE FLAP FAILURE USING A MULTI-INSTITUTIONAL DATABASE

There are numerous factors that may contribute to microvascular free flap failure. Although technical issues are dominant factors, patient and clinical characteristics are also contributory. The aim of this study was to investigate non‐technical variables associated with microsurgical free flap failure using a multi‐institutional dataset.

9-Cis Retinoic Acid Promotes Lymphangiogenesis and Enhances Lymphatic Vessel Regeneration: Therapeutic Implications of 9-Cis Retinoic Acid for Secondary Lymphedema

Background— The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction caused by genetic defects, or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swelling often associated with fibrosis and recurrent infections with no available cures to date. In this study, retinoic acids (RAs) were determined to be a potent therapeutic agent that is immediately applicable to reduce secondary lymphedema. Methods and Results— We report that RAs promote proliferation, migration, and tube formation of cultured lymphatic endothelial cells by activating fibroblast growth factor receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27Kip1, p57Kip2, and the aurora kinases through both an Akt-mediated nongenomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRA was found to activate in vivo lymphangiogenesis in animals in mouse trachea, Matrigel plug, and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration. Conclusion— These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients. # Clinical Perspective {#article-title-36}Background— The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction caused by genetic defects, or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swelling often associated with fibrosis and recurrent infections with no available cures to date. In this study, retinoic acids (RAs) were determined to be a potent therapeutic agent that is immediately applicable to reduce secondary lymphedema. Methods and Results— We report that RAs promote proliferation, migration, and tube formation of cultured lymphatic endothelial cells by activating fibroblast growth factor receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27Kip1, p57Kip2, and the aurora kinases through both an Akt-mediated nongenomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRA was found to activate in vivo lymphangiogenesis in animals in mouse trachea, Matrigel plug, and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration. Conclusion— These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients.

Ionizing Radiation of Mesenchymal Stem Cells Results in Diminution of the Precursor Pool and Limits Potential for Multilineage Differentiation

Background: Although ionizing radiation is an important treatment modality for a number of malignancies, it can be associated with significant morbidity. The exact mechanisms by which ionizing radiation results in cellular injury remain unknown. Mesenchymal stem cells give rise to a number of tissues including bone, fat, and cartilage and provide an excellent cellular model with which to evaluate the effects of ionizing radiation on cellular survival and function. Methods: Rat mesenchymal stem cells were irradiated with 0, 7, and 12 Gy of ionizing radiation and assessed for changes in growth, apoptosis, cell-cycle profile, senescence, differentiation, and gene expression. Results: Ionizing radiation resulted in a significant decrease in cellular proliferation because of increased apoptosis, G2 cell-cycle arrest, and premature senescence. In addition, ionizing radiation caused low-level spontaneous osteoblastic differentiation. Conversely, cellular differentiation in response to lineage-specific culture conditions for bone, fat, and cartilage was markedly decreased in irradiated cells, thereby demonstrating a deficit in the ability of irradiated mesenchymal stem cells to respond to environmental stimuli. Conclusions: Although the majority of mesenchymal stem cells survive injury from ionizing radiation, this injury results in a significant decrease in cellular proliferation. Furthermore, the differentiation potential of irradiated mesenchymal stem cells in response to environmental stimuli is markedly diminished. Thus, the negative effects of ionizing radiation may result from a decreased pool of progenitor cells with limited differentiation potential. Proposed radioprotection strategies aiming to reduce tissue injury should therefore evaluate not only cellular survival but also cellular function.

An Inducible Melanoma Model Implicates a Role for RAS in Tumor Maintenance and Angiogenesis

The established tumor is maintained through complex and poorly understood host-tumor interactions guiding processes such as angiogenesis. The numerous and diverse genetic alterations that accompany tumor genesis raises questions as to whether experimental cancer-promoting mutations remain relevant to tumor maintenance. Utilizing a new doxycycline-inducible H-RASV12G INK4a null mouse melanoma model, we have shown that melanoma genesis and maintenance are strictly dependent upon H-RASV12G expression. Withdrawal of doxycycline and H-RASV12G down-regulation resulted in clinical and histological regression of primary and explanted tumors. Moreover, the initial stages of regression were highlighted by dramatic activation of apoptosis in the tumor cells as well as host-derived endothelial cells. These data provide genetic evidence that H-RASV12G plays a critical role in tumor maintenance and tumor angiogenesis