Cell plasticity in wound healing: paracrine factors of M1/ M2 polarized macrophages influence the phenotypical state of dermal fibroblasts.

BACKGROUND: Macrophages and fibroblasts are two major players in tissue repair and fibrosis. Despite the relevance of macrophages and fibroblasts in tissue homeostasis, remarkably little is known whether macrophages are able to influence the properties of fibroblasts. Here we investigated the role of paracrine factors secreted by classically activated (M1) and alternatively activated (M2) human macrophages on human dermal fibroblasts (HDFs). RESULTS: HDFs stimulated with paracrine factors from M1 macrophages showed a 10 to > 100-fold increase in the expression of the inflammatory cytokines IL6, CCL2 and CCL7 and the matrix metalloproteinases MMP1 and MMP3. This indicates that factors produced by M1 macrophages induce a fibroblast phenotype with pro-inflammatory and extracellular matrix (ECM) degrading properties. HDFs stimulated with paracrine factors secreted by M2 macrophages displayed an increased proliferation rate. Interestingly, the M1-activated pro-inflammatory fibroblasts downregulated, after exposure to paracrine factors produced by M2 macrophages or non-conditioned media, the inflammatory markers as well as MMPs and upregulated their collagen production. CONCLUSIONS: Paracrine factors of M1 or M2 polarized macrophages induced different phenotypes of HDFs and the HDF phenotypes can in turn be reversed, pointing to a high dynamic plasticity of fibroblasts in the different phases of tissue repair.

Hemorrhagic shock-induced endothelial cell activation in a spontaneous breathing and a mechanical ventilation hemorrhagic shock model is induced by a proinflammatory response and not by hypoxia.

INTRODUCTION: The interaction between neutrophils and activated endothelium is essential for the development of multiple organ dysfunction in patients with hemorrhagic shock (HS). Mechanical ventilation frequently is used in patients with HS. The authors sought to investigate the consequences of mechanical ventilation of mice subjected to HS on microvascular endothelial activation in the lung and kidney. METHODS: Anesthetized wild type C57BL/6 male mice were subjected to controlled hemorrhage; subgroups of mice were mechanically ventilated during the HS insult. To study the effect of acute hypoxia on the mice, the animals were housed in hypoxic cages. Gene expression levels was assessed by quantitative real-time polymerase chain reaction. Protein expression was assessed by immunohistochemistry and enzyme-linked immunosorbent assay. RESULTS: Ninety minutes after the shock induction, a vascular bed-specific, heterogeneous proinflammatory endothelial activation represented by E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 expression was seen in kidney and lung. No differences in adhesion molecules between the spontaneously breathing and mechanically ventilated mice were found. Concentrations of the proinflammatory cytokines chemokine (C-X-C motif) ligand 1 (11.0-fold) and interleukin-6 (21.7-fold) were increased after 90 min of HS. Two hours of 6% oxygen did not induce the expression of E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 in the kidneys and the lung. CONCLUSIONS: Hemorrhagic shock leads to an early and reversible proinflammatory endothelial activation in kidney and lung. HS-induced endothelial activation is not changed by mechanical ventilation during the shock phase. Hypoxia alone does not lead to endothelial activation. The observed proinflammatory endothelial activation is mostly ischemia- or reperfusion-dependent and not related to hypoxia.

Dependence of neovascularization mechanisms on the molecular microenvironment.

In vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of bone marrow-derived endothelial cells (BMDECs), or both. Here we investigated the relative contribution of these 2 mechanisms to neovascularization in a mouse model of a foreign body reaction (FBR) to subcutaneously implanted Dacron and in hind limb ischemia (HLI) in relation to the molecular microenvironment at these neovascularization sites. Neovascularization was studied in C57Bl/6 mice reconstituted with enhanced green fluorescent protein (EGFP) transgenic bone marrow. Sprouting angiogenesis, detected using nuclear incorporation of bromodeoxyuridine in endothelial cells was present in both models, whereas vascular incorporation of EGFP(+) BMDECs was restricted to HLI. In HLI, the presence of a pro-angiogenic molecular microenvironment comprising vascular endothelial growth factor, fibroblast growth factor 2, and granulocyte colony-stimulating factor corroborated the importance of these factors for vascular BMDEC incorporation, whereas this microenvironment was absent in FBR. Enhanced mobilization of BMDECs by granulocyte-macrophage colony-stimulating factor administration or by combining HLI and FBR with Dacron did not induce incorporation of BMDECs in FBR neovessels. We conclude that the efficacy of BMDEC-based therapy is not generally warranted, but it depends on the molecular microenvironment in the targeted tissue.

17beta-estradiol and progesterone do not influence the production of cytokines from lipopolysaccharide-stimulated monocytes in humans.

OBJECTIVE: To test whether 17beta-estradiol or progesterone influence the cytokine productive capacity of lipopolysaccharide (LPS)-stimulated monocytes in humans. DESIGN: Prospective study. SETTING: Academic research institution. PATIENT(S): Seven women in the luteal phase of a normal ovarian cycle, 13 oral contraceptive users, 10 men, and 10 postmenopausal women. INTERVENTION(S): Blood samples collected from women in the luteal phase and from oral contraceptive users were used to study the in vivo effect of 17beta-estradiol and progesterone on monocyte cytokine production. Blood samples collected from men and postmenopausal women were used for in vitro incubation with 17beta-estradiol and progesterone. MAIN OUTCOME MEASURE(S): The percentage of monocytes producing tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) after in vitro LPS-stimulation was determined. RESULT(S): No in vivo relation in the productive capacities of LPS-stimulated monocytes in the luteal phase of the ovarian cycle was found between progesterone and TNF-alpha or IL-1beta; or between 17beta-estradiol and TNF-alpha or IL-1beta. Moreover, the production of TNF-alpha and IL-1beta by LPS-stimulated monocytes did not vary between periods of oral contraceptive use and nonuse. The production of TNF-alpha and IL-1beta by LPS-stimulated monocytes in the blood of men and postmenopausal women in vitro was not influenced by incubation with different concentrations of 17beta-estradiol or progesterone. CONCLUSION(S): We could not find evidence for a causal relationship between 17beta-estradiol or progesterone and TNF-alpha- or IL-1beta-production. We conclude that 17beta-estradiol and progesterone do not influence the cytokine-production capacity of LPS-stimulated monocytes in humans.

Beneficial effects of an alternating high- fat dietary regimen on systemic insulin resistance, hepatic and renal inflammation and renal function.

BACKGROUND: An Alternating high- cholesterol dietary regimen has proven to be beneficial when compared to daily high- cholesterol feeding. In the current study we explored whether the same strategy is applicable to a high- fat dietary regimen. OBJECTIVE: To investigate whether an alternating high- fat dietary regimen can effectively diminish insulin resistance, hepatic and renal inflammation and renal dysfunction as compared to a continuous high- fat diet. DESIGN: Four groups of male ApoE*3Leiden mice (n=15) were exposed to different diet regimens for 20 weeks as follows: Group 1: low- fat diet (10 kcal% fat); Group 2: intermediate- fat diet (25 kcal% fat); Group 3: high- fat diet (45 kcal% fat) and Group 4: alternating- fat diet (10 kcal% fat for 4 days and 45 kcal% fat for 3 days in a week). RESULTS: Compared to high fat diet feeding, the alternating and intermediate- fat diet groups had reduced body weight gain and did not develop insulin resistance or albuminuria. In addition, in the alternating and intermediate- fat diet groups, parameters of tissue inflammation were markedly reduced compared to high fat diet fed mice. CONCLUSION: Both alternating and intermediate- fat feeding were beneficial in terms of reducing body weight gain, insulin resistance, hepatic and renal inflammation and renal dysfunction. Thus beneficial effects of alternating feeding regimens on cardiometabolic risk factors are not only applicable for cholesterol containing diets but can be extended to diets high in fat content.

Circulating CD34+ progenitor cells modulate host angiogenesis and inflammation in vivo.

Within the phenotypically and functionally heterogeneous group of circulating progenitor cells (CPC), a subclass of cells with vascular repair potential have been identified. These CPC are detected and isolated based on single or combined expression of CD34, CD133 and VEGFR-2, and referred to as endothelial progenitor cells. Here we asked whether CPC subsets defined by single expression of these markers exhibit functional heterogeneity. As functional parameters, we chose the capacity of CPC to differentiate into endothelial cells. Moreover, we studied their role in remodeling by recruitment of inflammatory cells, an aspect that has been little explored. We established an in vivo model in which the intrinsic functional capacity of these human CPC subsets was studied. Human CD34+ CPC, but not CD133+ or VEGFR-2+ CPC, seeded in Matrigel pellets and transplanted subcutaneously in a nude mouse host, contributed little to donor-derived neovascularization. However, host angiogenesis in the Matrigel implant, as demonstrated by the presence of capillaries containing erythrocytes and expressing mouse CD31, was strong in response to implantation of human CD34+ CPC and significantly lower in response to the other two CPC subsets. Moreover, the CD34+ CPC subset was significantly superior to CD133+ CPC and VEGFR-2+ CPC in the recruitment of host monocytes/macrophages. These three CPC populations were further dissected into seven discrete subsets, based on three-parameter flow cytometry analysis of combined expression patterns of CD34, CD133 and VEGFR-2. In conclusion, in our system, CD34+ CPC contribute marginally to neovascularization by differentiation but are potent regulators of the host angiogenic and pro-inflammatory response, suggesting a possible role for these cells in the remodeling of vascular lesions.

Methylenetetrahydrofolate reductase (MTHFR) and susceptibility for (pre)neoplastic cervical disease.

Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme regulating the metabolism of folate and methionine. The potential influence of MTHFR activity on DNA methylation and on the availability of uridylates and thymidylates for DNA synthesis and repair presents MTHFR as a candidate for being a cancer-predisposing gene. In the present study, we have examined a large study population to determine whether the C677T polymorphism at the MTHFR locus affects susceptibility for cervical cancer or its precursor, cervical intraepithelial neoplasia (CIN). In addition, we have investigated whether this polymorphism is causal, and not merely associated, by typing microsatellite markers in the region surrounding the MTHFR gene. A total of 311 CIN and 695 cervical cancer patients and 115 family-based and 586 unrelated controls was analysed. Association analysis showed a decreased cervical cancer risk for individuals heterozygous or homozygous for the T-allele, both for squamous cell carcinoma (heterozygous odds ration [OR] 0.66 [0.51-0.86]; homozygous OR 0.76 [0.49-1.16]) and adenocarcinoma (heterozygous OR 0.71 [0.49-1.03]; homozygous OR 0.34 [0.14-0.81]). No difference was found for high grade CIN (heterozygous OR 1.03 [0.76-1.40]; homozygous OR 0.91 [0.54-1.55]). A microsatellite haplotype containing the C allele was associated with an increased risk for cervical cancer and CIN (both among squamous cell carcinomas, adenocarcinomas and CIN II-III; OR = 2.61 [1.59-4.27]). Our study thus lends further support to the hypothesis that the MTHFR C677T polymorphism is involved in susceptibility cervical cancer but also illustrates that, despite the large sample size analysed, still larger studies are needed to establish fully the nature of this association.

Gender difference in the non-specific and specific immune response in humans.

PROBLEM: The purpose of this present ex vivo study is to get insight in the sex differences of the basic non-specific and specific immune response. METHOD OF STUDY: Intracellular types 1 and 2 cytokine production by stimulated male and female lymphocytes and monocytes in a whole blood preparation was measured by flow cytometry. RESULTS: Increased percentage interleukin (IL)-12, IL-1 beta and tumor necrosis factor (TNF)-alpha producing monocytes and decreased percentage IL-2 producing lymphocytes, i.e. type 1 cytokine, were found in men as compared with women. CONCLUSION: These results suggest a gender difference in the balance between the specific and non-specific immune response, i.e. a more profound and higher state of excitation of the non-specific immune response and relative suppression of the cellular immune response of the specific immune system in men as compared with women.