Joshua Tam

Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature

Vascular tissue engineering requires a ready source of endothelial cells and perivascular cells. Here, we evaluated human bone marrow-derived mesenchymal stem cells (hMSCs) for use as vascular progenitor cells in tissue engineering and regenerative medicine. hMSCs expressed a panel of smooth muscle markers in vitro including the cardiac/smooth muscle-specific transcription coactivator, myocardin. Cell-cell contact between endothelial cells and hMSCs up-regulated the transcription of myocardin. hMSCs efficiently stabilized nascent blood vessels in vivo by functioning as perivascular precursor cells. The engineered blood vessels derived from human umbilical cord vein endothelial cells and hMSCs remained stable and functional for more than 130 days in vivo. On the other hand, we could not detect differentiation of hMSCs to endothelial cells in vitro, and hMSCs by themselves could not form conduit for blood flow in vivo. Similar to normal perivascular cells, hMSC-derived perivascular cells contracted in response to endothelin-1 in vivo. In conclusion, hMSCs are perivascular cell precursors and may serve as an attractive source of cells for use in vascular tissue engineering and for the study of perivascular cell differentiation.

Paracrine Regulation of Angiogenesis and Adipocyte Differentiation During In Vivo Adipogenesis

Abstract— With an increasing incidence of obesity worldwide, rational strategies are needed to control adipogenesis. Growth of any tissue requires the formation of a functional and mature vasculature. To gain mechanistic insight into the link between active adipogenesis and angiogenesis, we developed a model to visualize noninvasively and in real time both angiogenesis and adipogenesis using intravital microscopy. Implanted murine preadipocytes induced vigorous angiogenesis and formed fat pads in a mouse dorsal skin-fold chamber. The newly formed vessels subsequently remodeled into a mature network consisting of arterioles, capillaries, and venules, whereas the preadipocytes differentiated into adipocytes as confirmed by increased aP2 expression. Inhibition of adipocyte differentiation by transfection of preadipocytes with a peroxisome proliferator-activated receptor &ggr; dominant-negative construct not only abrogated fat tissue formation but also reduced angiogenesis. Surprisingly, inhibition of angiogenesis by vascular endothelial growth factor receptor-2 (VEGFR2) blocking antibody not only reduced angiogenesis and tissue growth but also inhibited preadipocyte differentiation. We found that part of this inhibition stems from the paracrine interaction between endothelial cells and preadipocytes and that VEGF–VEGFR2 signaling in endothelial cells, but not preadipocytes, mediates this process. These findings reveal a reciprocal regulation of adipogenesis and angiogenesis, and suggest that blockade of VEGF signaling can inhibit in vivo adipose tissue formation. The full text of this article is available online at http://www.circresaha.org.

Blockade of VEGFR2 and Not VEGFR1 Can Limit Diet-Induced Fat Tissue Expansion: Role of Local versus Bone Marrow-Derived Endothelial Cells

Background We investigated if new vessel formation in fat involves the contribution of local tissue-derived endothelial cells (i.e., angiogenesis) or bone marrow-derived cells (BMDCs, i.e. vasculogenesis) and if antiangiogenic treatment by blockade of vascular endothelial growth factor (VEGF) receptors can prevent diet-induced obesity (DIO). Methodology/Principal Findings We performed restorative bone marrow transplantation into wild-type mice using transgenic mice expressing green fluorescent protein (GFP) constitutively (driven by β-actin promoter) or selectively in endothelial cells (under Tie2 promoter activation) as donors. The presence of donor BMDCs in recipient mice was investigated in fat tissue vessels after DIO using in vivo and ex vivo fluorescence microscopy. We investigated the roles of VEGF receptors 1 and 2 (VEGFR1/VEGFR2) by inducing DIO in mice and treating them with blocking monoclonal antibodies. We found only marginal (less than 1%) incorporation of BMDCs in fat vessels during DIO. When angiogenesis was inhibited by blocking VEGFR2 in mice with DIO, treated mice had significantly lower body weights than control animals. In contrast, blocking VEGFR1 had no discernable effect on the weight gain during DIO. Conclusions/Significance Formation of new vessels in fat tissues during DIO is largely due to angiogenesis rather than de novo vasculogenesis. Antiangiogenic treatment by blockade of VEGFR2 but not VEGFR1 may limit adipose tissue expansion.

Enhanced uptake and photoactivation of topical methyl aminolevulinate after fractional CO2 laser pretreatment.

Photodynamic therapy (PDT) of thick skin lesions is limited by topical drug uptake. Ablative fractional resurfacing (AFR) creates vertical channels that may facilitate topical PDT drug penetration and improve PDT‐response in deep skin layers. The purpose of this study was to evaluate whether pre‐treating the skin with AFR before topically applied methyl aminolevulinate (MAL) could enable a deep PDT‐response.

Fractional laser-assisted delivery of methyl aminolevulinate: Impact of laser channel depth and incubation time†

Pretreatment of skin with ablative fractional lasers (AFXL) enhances the uptake of topical photosensitizers used in photodynamic therapy (PDT). Distribution of photosensitizer into skin layers may depend on depth of laser channels and incubation time. This study evaluates whether depth of intradermal laser channels and incubation time may affect AFXL‐assisted delivery of methyl aminolevulinate (MAL).

Pretreatment with ablative fractional laser changes kinetics and biodistribution of topical 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL)

5‐Aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) are porphyrin precursors used topically for photodynamic therapy (PDT). Previous studies have established that ablative fractional laser (AFXL) increases topical drug uptake. We evaluated kinetics and biodistribution of ALA‐ and MAL‐induced porphyrins on intact and disrupted skin due to AFXL.

Paradoxical Effects of PDGF-BB Overexpression in Endothelial Cells on Engineered Blood Vessels In Vivo

Therapeutic revascularization with either exogenous angiogenic growth factors or vascular cells has yet to demonstrate efficacy in the clinic. Injection of angiogenic growth factors often produces unstable and abnormal blood vessels. Blood vascular networks derived from implanted endothelial cells persist only transiently due to the insufficient recruitment of perivascular cells. We hypothesize that a combination of the two approaches may act synergistically to yield a better result. To enhance the recruitment of perivascular cells, human umbilical vein endothelial cells were genetically modified to overexpress platelet-derived growth factor (PDGF)-BB. PDGF-BB overexpression promoted both proliferation and migration of perivascular precursor cells (10T1/2 cells) in vitro. When mock-infected endothelial cells were implanted alone in vivo, they formed transient blood vascular networks that regressed by day 30. PDGF-BB overexpression enhanced the survival of endothelial cells in vivo. However, the PDGF-BB-expressing vessel network failed to establish patent blood flow. Co-implantation of PDGF-BB-overexpressing endothelial cells with 10T1/2 cells paradoxically resulted in the rapid regression of the vascular networks in vivo. PDGF-BB stimulated the expression of both chemokine (C-C motif) ligand 2 (CCL2) and CCL7 in 10T1/2 cells and led to the increased accumulation of macrophages in vivo. These results suggest a potential negative interaction between angiogenic growth factors and vascular cells; their use in combination should be carefully tested in vivo for such opposing effects.

Lack of lymphatic vessel phenotype in LYVE-1/CD44 double knockout mice.

Lymphatic vessels play a key role in maintaining tissue‐fluid homeostasis, immune surveillance and metastasis. The hyaluronan receptor, LYVE‐1, is widely used as a molecular marker for adult and embryonic lymphatic endothelium, but its physiological functions have not yet been established in vivo. In agreement with a recent report, LYVE‐1−/− mice, which are healthy and fertile, do not display any defects related to congenital abnormalities of the lymphatic system. One hypothesis for the absence of a phenotype in LYVE‐1 null mice is that other hyaluronan receptors, such as CD44, may compensate for LYVE‐1. To test this hypothesis, we created LYVE‐1/CD44 double knockout mice with appropriate littermate controls. Lymphatic vessel structure and function, as determined by histological methods and intravital microscopy, show that LYVE‐1−/−, CD44−/− and LYVE‐1−/−/CD44−/− mice are indistinguishable from wild‐type mice under normal conditions. Furthermore, resolution of carrageenan‐induced paw edema is comparable in all genotypes. However, LYVE‐1−/−/CD44−/− mice exhibit increased edema formation in a carrageenan‐induced paw inflammation model compared to wild‐type mice, but not to LYVE−/− or CD44−/− mice. These data suggest that LYVE‐1 and CD44 are not required for the formation or function of lymphatics, but do not rule out a role for LYVE‐1 in inflammation. J. Cell. Physiol. 219: 430–437, 2009.

Fractional Skin Harvesting: Autologous Skin Grafting without Donor-site Morbidity

Background: Conventional autologous skin grafts are associated with significant donor-site morbidity. This study was conducted to determine feasibility, safety, and efficacy of a new strategy for skin grafting based on harvesting small columns of full-thickness skin with minimal donor-site morbidity. Methods: The swine model was used for this study. Hundreds of full-thickness columns of skin tissue (~700 µm diameter) were harvested using a custom-made harvesting device, and then applied directly to excisional skin wounds. Healing in donor and graft sites was evaluated over 3 months by digital photographic measurement of wound size and blinded, computer-aided evaluation of histological features and compared with control wounds that healed by secondary intention or with conventional split-thickness skin grafts (STSG). Results: After harvesting hundreds of skin columns, the donor sites healed rapidly without scarring. These sites reepithelialized within days and were grossly and histologically indistinguishable from normal skin within 7 weeks. By contrast, STSG donor sites required 2 weeks for reepithelialization and retained scar-like characteristics in epidermal and dermal architecture throughout the experiment. Wounds grafted with skin columns resulted in accelerated reepithelialization compared with ungrafted wounds while avoiding the “fish-net” patterning caused by STSG. Conclusion: Full-thickness columns of skin can be harvested in large quantities with negligible long-term donor-site morbidity, and these columns can be applied directly to skin wounds to enhance wound healing.

Selective Cryolysis of Sebaceous Glands.

Acne vulgaris is a nearly universal cutaneous inflammatory disease. Excess sebum production is an integral part of disease pathogenesis. Medical therapies that reduce sebum excretion result in clinical improvement of acne. Given the preferential susceptibility of lipid-containing cells to cold, we investigated the hypothesis that controlled local skin cooling causes preferential injury to sebaceous glands, in murine and swine models using a range of temperatures as low as -10 °C, and then on the backs of human subjects. In mouse ears, peak histologic damage occurred 72 hours after treatment; eosinophilic necrotic plugs formed within sebaceous glands, and the number of glands was significantly reduced up to 1 week post treatment. Cooling disrupted sebocyte cell membranes, alkaline phosphatase activity, and significantly reduced sebocyte lipid content. In human volunteers, cooling damaged sebaceous glands and reduced sebum output for 2 weeks, with minimal injury to surrounding tissues. Selective cryolysis of sebaceous glands is achievable through brief, non-invasive skin cooling, suggesting that controlled cooling could be developed as an effective treatment for acne vulgaris.