Impaired wound healing is a common complication of diabetes. Although it is well known that both macrophages and blood vessels are critical to wound repair, the role of wound-associated lymphatic vessels has not been well investigated. We report that both the presence of activated macrophages and the formation of lymphatic vessels are rate-limiting to the healing of diabetic wounds. We have previously shown that macrophages contribute to the lymphatic vessels that form during the acute phase of corneal wound healing. We now demonstrate that this is a general phenomenon; cells that co-stain for the macrophage marker F4/80 and the lymphatic markers LYVE-1 (lymphatic vascular endothelium hyaluronate receptor) and podoplanin contribute to lymphatic vessels in full-thickness wounds. LYVE-1-positive lymphatic vessels and CD31-positive blood vessels were significantly reduced in corneal wound healing in diabetic mice (db/db) (P < 0.02) compared with control (db/؉) mice. Glucose treatment of control macrophages led to the down-regulation of the lymphaticspecific receptor VEGFR3 and its ligands, vascular endothelial growth factor-C and -D (VEGF-C, -D). Interleukin-1␤ stimulation rescued diabetic macrophage function; application of interleukin-1␤-treated db/db-derived macrophages to wounds in db/db mice induced lymphatic vessel formation and accelerated wound healing. These observations suggest a potential therapeutic approach for healing wounds in diabetic patients.

METHODS Migration AssaysMigration assays were performed as described (3)(4)(5). Briefly, 16 h before the assay, 80% confluent 75 cm 2 flasks (Corning Costar) of human microvessel endothelial cells (HMVEC; Cambrex, Walkersville, MD), human coronary artery endothelial cells (HCAEC; Cambrex), human umbilical artery endothelial cells (HUAEC; Promocell, Heidelburg, Germany), or human umbilical vein endothelial cells (HUVEC; Promocell), were washed with Hank's Balanced Salt Solution (HBSS, Invitrogen) and serum-starved overnight in endothelial basal media (EBM-2, Cambrex) with 0.1% fatty-acid-free BSA (Sigma) and 0.5% fetal calf serum (FCS, Hyclone). The following day cells were lifted with Trypsin/EDTA solution (Promocell), mixed with an equal volume Trypsin Neutralization Solution (Promocell), and washed 3 times in migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS). Cells were resuspended at a density of 1.5×10 6 cells/ml and were allowed to recover for 1 h at 37°C (5% CO 2 ). 3.75 × 10 4 cells were plated into each well of a 48-well Boyden chamber apparatus (NeuroProbe, Cabin John, MD), and the wells were overlayed with an 8 μm pore polycarbonate membrane (NeuroProbe) that had been previously coated with 50 μg/ml human fibronectin (Biomedical Technologies, Inc., Stoughton, MA). Experiments performed with membranes coated with acetylated 1% gelatin from porcine skin (Sigma, St. Louis, MO) gave similar results. The apparatus was assembled and stored inverted at 37°C (5% CO 2 ) for 2 h. The apparatus was then re-inverted and 52 μl of purified chemoattractants [murine netrin-1 (R&D Systems, Minneapolis, MN), chicken netrin-2 (R&D Systems), murine netrin-4 (R&D Systems), murine netrin-G1a (R&D Systems), human VEGF 165 (R&D Systems), or control/ migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS) were added to the upper chambers, and the migration was allowed to proceed for 2 h at 37°C (5% CO 2 ). The membranes were then removed, fixed in methanol, stained with a Hema 3 stain set (Fisher Scientific, Pittsburgh, PA), and placed (migrated-side down) onto 50 × 75 mm glass slides. Before 90% mounting medium (in xylenes) and coverslips were applied, the non-migrated cells were removed from the exposed (non-migrated) side of the membrane with a moistened swab. Cells present on the migrated side of the membrane were manually counted (three random 200× fields per well), and data points for each experiment represent the average number of migrated cells from six separate wells (three 200× fields counted per well).Another method was employed in a separate laboratory to evaluate the effects of the netrins on mouse (MS1) endothelial cells (ATCC, Manassas, VA) using a modified Boyden chamber assay as described previously (6). Briefly, a 5 μm-polycarbonate filter (Poretics) was placed between upper and lower chamber. Cell suspensions (5×10 4 cells/well) were placed in the upper chamber, and the lower chamber was filled with serum-free medium containing

Sonic hedgehog (Shh) is a crucial regulator of organ development during embryogenesis. We investigated whether intramyocardial gene transfer of naked DNA encoding human Shh (phShh) could promote a favorable effect on recovery from acute and chronic myocardial ischemia in adult animals, not only by promoting neovascularization, but by broader effects, consistent with the role of this morphogen in embryogenesis. After Shh gene transfer, the hedgehog pathway was upregulated in mammalian fibroblasts and cardiomyocytes. This resulted in preservation of left ventricular function in both acute and chronic myocardial ischemia by enhanced neovascularization, and reduced fibrosis and cardiac apoptosis. Shh gene transfer also enhanced the contribution of bone marrow-derived endothelial progenitor cells to myocardial neovascularization. These data suggest that Shh gene therapy may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by triggering expression of multiple trophic factors and engendering tissue repair in the adult heart.

Background— Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial-mesenchymal interaction during embryonic development. Recent observations indicate that exogenous administration of Shh can induce angiogenesis and may accelerate repair of ischemic myocardium and skeletal muscle. Because angiogenesis plays a pivotal role in wound repair, we hypothesized that activation of the hedgehog pathway may promote a favorable effect on microvascular remodeling during cutaneous wound healing and thereby accelerate wound closure. Because diabetes is associated with impaired wound healing, we tested this hypothesis in a diabetic model of cutaneous wound repair. Methods and Results— In Ptc1-LacZ mice, cutaneous injury resulted in LacZ expression, indicating that expression of the Shh receptor Patched was induced and therefore that the Shh signaling pathway was intact postnatally and upregulated in the process of wound repair. In diabetic mice, topical gene therapy with the use of naked DNA encoding for Shh resulted in significant local gene expression and acceleration of wound recovery. The acceleration in wound healing was notable for increased wound vascularity. In bone marrow transplantation models, the enhanced vascularity of the wound was shown to be mediated, at least in part, by enhanced recruitment of bone marrow–derived endothelial progenitor cells. In vitro, Shh promoted production of angiogenic cytokines from fibroblasts as well as proliferation of dermal fibroblasts. Furthermore, Shh directly promoted endothelial progenitor cell proliferation, migration, adhesion, and tube formation. Conclusions— These findings suggest that a simple strategy of topically applied Shh gene therapy may have significant therapeutic potential for enhanced wound healing in patients with impaired microcirculation such as occurs in diabetes. (Circulation. 2006;113:2413-2424.)

Abstract-Delayed reendothelialization contributes to restenosis after angioplasty and stenting in diabetes. Prior data have shown that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to endothelial recovery after arterial injury. We investigated the hypothesis that the EPC contribution to reendothelialization may be impaired in diabetes, resulting in delayed reendothelialization. Reendothelialization was significantly reduced in diabetic mice compared with nondiabetic mice in a wire-induced carotid denudation model. The EPC contribution to neoendothelium was significantly reduced in Tie2/LacZ BM-transplanted diabetic versus nondiabetic mice. BM from diabetic and nondiabetic mice was transplanted into nondiabetic mice, revealing that reendothelialization was impaired in the recipients of diabetic BM. To examine the relative roles of denuded artery versus EPCs in diabetes, we injected diabetic and nondiabetic EPCs intravenously after arterial injury in diabetic and nondiabetic mice. Diabetic EPCs recruitment to the neoendothelium was significantly reduced, regardless of the diabetic status of the recipient mice. In vitro, diabetic EPCs exhibited decreased migration and adhesion activities. Vascular endothelial growth factor and endothelial NO synthase expressions were also significantly reduced in diabetic EPCs. Notably, thrombospondin-1 mRNA expression was significantly upregulated in diabetic EPCs, associating with the decreased EPC adhesion activity in vitro and in vivo.

Inflammation plays an essential role in vascular injury and repair. Mononuclear phagocytes are important contributors in these processes, in part, via adhesive interactions and secretion of proinflammatory cytokines. The antiinflammatory cytokine interleukin (IL)-10 suppresses such responses via deactivation of monocytes/macrophages and repression of inflammatory cytokine expression. The mechanisms of IL-10's suppressive action are, however, incompletely characterized. Here, we report that systemic IL-10 treatment after carotid artery denudation in mice blunts inflammatory cell infiltration and arterial tumor necrosis factor (TNF) expression. At the molecular level, in a human monocytic cell line, U937 IL-10 suppressed LPS-induced mRNA expression of a number of inflammatory cytokines, mainly via posttranscriptional mRNA destabilization. Detailed studies on IL-10 regulation of TNF-alpha mRNA expression identified AU-rich elements (ARE) in the 3' untranslated region as a necessary determinant of IL-10-mediated TNF-alpha mRNA destabilization. IL-10 sensitivity to TNF depends on the ability of IL-10 to inhibit the expression and mRNA-stabilizing protein HuR and via IL-10 mediated repression of p38 mitogen-activated protein (MAP) kinase activation. Because IL-10 function and signaling are important components for control of inflammatory responses, these results may provide insights necessary to develop strategies for modulating vascular repair and other accelerated arteriopathies, including transplant vasculopathy and vein graft hyperplasia.

Background-Estradiol (E 2 ) modulates the kinetics of circulating endothelial progenitor cells (EPCs) and favorably affects neovascularization after ischemic injury. However, the roles of estrogen receptors ␣ (ER␣) and ␤ (ER␤) in EPC biology are largely unknown. Methods and Results-In response to E 2 , migration, tube formation, adhesion, and estrogen-responsive elementdependent gene transcription activities were severely impaired in EPCs obtained from ER␣-knockout mice (ER␣KO) and moderately impaired in ER␤KO EPCs.

Podoplanin (PDPN) is a transmembrane receptor glycoprotein that is upregulated on transformed cells, cancer associated fibroblasts and inflammatory macrophages that contribute to cancer progression. In particular, PDPN increases tumor cell clonal capacity, epithelial mesenchymal transition, migration, invasion, metastasis and inflammation. Antibodies, CAR‐T cells, biologics and synthetic compounds that target PDPN can inhibit cancer progression and septic inflammation in preclinical models. This review describes recent advances in how PDPN may be used as a biomarker and therapeutic target for many types of cancer, including glioma, squamous cell carcinoma, mesothelioma and melanoma.