Diabetes is characterized by poor circulation and impaired angiogenesis, which appear to contribute to the frequent skin lesions and poor wound healing common in diabetic patients. Therapies to improve circulation commonly improve wound healing in diabetic patients. Administration of circulating CD34+ cells, cells that can function as endothelial cell progenitors, accelerates blood flow restoration to ischemic limbs of diabetic mice. We have investigated the potential of these cells to accelerate revascularization and healing in full-thickness skin wounds of hypoinsulinemic (streptozotocin-treated) diabetic mice. Wounds were injected with human CD34+ or CD34– peripheral blood mononuclear cells or no cells, and analyzed for vascularity and healing at various times thereafter. Treatment with CD34+ enriched cells decreased wound size by 4 days after treatment, accelerated epidermal healing, and rapidly and dramatically accelerated revascularization of the wounds compared to controls. Initially increased vascularization was mediated principally by an increase in vessel diameter, but later, both an increase in vascular size and number were observed. These findings indicate that blood-derived progenitors may have therapeutic potential in the treatment of skin lesions in the setting of diabetes, and give insights into how bone marrow cells exert their effects on neovascularization.

Abstract-Bone marrow stem cells participate in tissue repair processes and may have roles in skin wound repair. Diabetes is characterized by delayed and poor wound healing, and type 1 diabetes seems to lead to stem cell dysfunction. Hence, stem cell dysfunction could contribute to poor healing, and stem cell-based therapies may be efficacious in diabetic wounds. We investigated the potential of exogenous stem cells to promote skin healing and possible effects of type 2 diabetes on stem cell function. Mouse bone marrow cells from nondiabetic and diabetic mice were enriched for putative stem cells and injected under skin wounds of nondiabetic or type 2 diabetic Lepr db mice. Using histology and morphometry, vascularization and healing in treated and untreated mice were analyzed. We anticipated a correlation between improved wound healing and vascularization, because therapies that increase tissue vascularization tend to enhance wound healing. Our data indicate that exogenous nondiabetic bone marrow-derived cells increase vascularization and improve wound healing in Lepr db mice but have little effect on nondiabetic controls. In contrast, Lepr db -derived marrow cells inhibit vascularization but promote wound healing in Lepr db mice. Thus, adult stem cell function may be impaired by type 2 diabetes; the ability to promote vascularization and wound healing are distinct functions of bone marrow cells; and neovascularization and wound healing may not be tightly coupled. Additionally, we observed little incorporation of injected cells into wound structures, suggesting that improved healing is mediated through mechanisms other than direct differentiation and incorporation of the cells.

Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive, multisystem disorder presenting in childhood with intermittent neutropenia and pancreatic insufficiency. It is characterized by recurrent infections independent of neutropenia, suggesting a functional neutrophil defect. While mutations at a single gene locus (SBDS) appear to be responsible for SDS in a majority of patients, the function of that gene and a specific defect in SDS neutrophil behavior have not been elucidated. Therefore, employing 2D and 3D computer-assisted motion analysis systems, we have analyzed the basic motile behavior and chemotactic responsiveness of individual polymorphonuclear leukocytes (PMNs) of 14 clinically diagnosed SDS patients. It is demonstrated that the basic motile behavior of SDS PMNs is normal in the absence of chemoattractant, that SDS PMNs respond normally to increasing and decreasing temporal gradients of the chemoattractant fMLP, and that SDS PMNs exhibit a normal chemokinetic response to a spatial gradient of fMLP. fMLP receptors were also distributed uniformly through the plasma membrane of SDS PMNs as in control PMNs. SDS PMNs, however, were incapable of orienting in and chemotaxing up a spatial gradient of fMLP. This unique defect in orientation was manifested by the PMNs of every SDS patient tested. The PMNs of an SDS patient who had received an allogenic hematopoietic stem cell transplant, as well as PMNs from a cystic fibrosis patient, oriented normally. These results suggest that the defect in SDS PMNs is in a specific pathway emanating from the fMLP receptor that is involved exclusively in regulating orientation in response to a spatial gradient of fMLP. This pathway must function in parallel with additional pathways, intact in SDS patients, that emanate from the fMLP receptor and regulate responses to temporal rather than spatial changes in receptor occupancy.

To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC ؊ , in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC ؊ cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC ؊ cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC ؊ cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC ؊ cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA ؊ /myoF ؊ myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC ؊ mutant are the immediate result of the absence of RasC function.The Ras GTPases function as molecular switches in the regulation of a variety of responses to extracellular signals (3,21,35,36). Dictyostelium discoideum, like higher eukaryotes, contains a number of Ras GTPases (6,21,22). Because of its attributes, Dictyostelium provides a unique experimental system for exploring the roles played by the Ras GTPases in cell motility and chemotaxis (6,21,49). First, because it is haploid, null mutations are readily generated and rescued (16,18). Second, because the behavior of Dictyostelium amoebae in buffer and in response to the temporal, spatial, and concentration components of the natural chemotactic wave has been characterized in detail by computer-assisted methods (33, 34), a unique contextual framework exists for identifying specific behavioral defects manifested in mutants (29) and for deducing from them the specific role played by mutated genes in motility and/or chemotaxis (4,8,10,42,45,50,51).In a previous study, it was demonstrated that rasC Ϫ cells could not progress through the early stages of development or form aggregates unless they were pulsed with the chemoattractant cyclic AMP (cAMP), indicating that RasC was necessary for signaling (20). rasC Ϫ cells artificially pulsed with cAMP were then capable of forming aggregates on filter pads. When mixed with a majority of normal cells, they could also enter aggr...

Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA ؊ cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA ؊ cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA ؊ cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.When Dictyostelium amoebae deplete their environment of nutrients, they aggregate and then, as a multicellular unit, undergo morphogenesis to generate a sporangium composed of a stalk supporting a cap containing spores (11). During aggregation, cells both relay the chemoattractant cyclic AMP (cAMP) through the population as a series of outwardly moving, nondissipating waves (38) and respond to the information in the waves by surging in a pulsatile manner toward the original aggregation center (1, 48).Pitt et al. (21) first identified and characterized the gene acaA, which encodes the adenylyl cyclase that produces the extracellular cAMP that serves as the chemotactic signal. Cells of the null mutant acaA Ϫ were incapable of aggregating because they could not release a chemotactic signal (21). Pitt et al. (21) found that if acaA Ϫ cells were induced to undergo early development by pulsing them experimentally with extracellular cAMP, they were then capable of undergoing chemotaxis in an experimentally generated spatial gradient of cAMP. Furthermore, acaA Ϫ cells artificially pulsed with cAMP made fruiting bodies, albeit smaller than those of wild-type cells. These observations were consistent with a purely extracellular role for cAMP in chemotaxis. However, it was subsequently demonstrated that the null mutant of the intracellular phosphodiesterase RegA and the null mutant of the protein kinase A (PKA) regulatory subunit were defective in their responses to an increasing temporal gradie...