Eugénia Carvalho

Recent advances on the development of wound dressings for diabetic foot ulcer treatment—A review

Diabetic foot ulcers (DFUs) are a chronic, non-healing complication of diabetes that lead to high hospital costs and, in extreme cases, to amputation. Diabetic neuropathy, peripheral vascular disease, abnormal cellular and cytokine/chemokine activity are among the main factors that hinder diabetic wound repair. DFUs represent a current and important challenge in the development of novel and efficient wound dressings. In general, an ideal wound dressing should provide a moist wound environment, offer protection from secondary infections, remove wound exudate and promote tissue regeneration. However, no existing dressing fulfills all the requirements associated with DFU treatment and the choice of the correct dressing depends on the wound type and stage, injury extension, patient condition and the tissues involved. Currently, there are different types of commercially available wound dressings that can be used for DFU treatment which differ on their application modes, materials, shape and on the methods employed for production. Dressing materials can include natural, modified and synthetic polymers, as well as their mixtures or combinations, processed in the form of films, foams, hydrocolloids and hydrogels. Moreover, wound dressings may be employed as medicated systems, through the delivery of healing enhancers and therapeutic substances (drugs, growth factors, peptides, stem cells and/or other bioactive substances). This work reviews the state of the art and the most recent advances in the development of wound dressings for DFU treatment. Special emphasis is given to systems employing new polymeric biomaterials, and to the latest and innovative therapeutic strategies and delivery approaches.

Low cellular IRS 1 gene and protein expression predict insulin resistance and NIDDM.

We examined the gene and protein expression of IRS 1 (insulin receptor substrate 1) in adipocytes from two groups of healthy individuals with an increased propensity for non‐insulin‐dependent diabetes mellitus (NIDDM): those with two first‐degree relatives with diabetes and another group with massive obesity. A low expression of IRS 1(<50% of the matched control group) was seen in «30% of both groups and these individuals were characterized by insulin resistance and its hallmarks: higher levels of insulin, glucose, and triglycerides. Two individuals with previously unknown NIDDM were diagnosed and both had low IRS 1 expression. Low IRS 1 protein expression was associated with low mRNA levels but not with the common Gly972Arg polymorphism of the IRS 1 gene. Taken together, our present and previous findings show that a low expression of IRS 1 in fat cells predicts insulin resistance and NIDDM. Furthermore, they support the likelihood that an impaired transcriptional activation may play a key role in the pathogenesis of NIDDM.—Carvalho, E., Jansson, P.‐A., Axelsen, M., Eriksson, J. W., Huang, X., Groop, L., Rondinone, C., Sjostrom, L., Smith, U. Low cellular IRS 1 gene and protein expression predict insulin resistance and NIDDM. FASEB J. 13, 2173–2178 (1999)

A novel cellular marker of insulin resistance and early atherosclerosis in humans is related to impaired fat cell differentiation and low adiponectin

The epidemic increase in type 2 diabetes can be prevented only if markers of risk can be identified and used for early intervention. We examined the clinical phenotype of individuals characterized by normal or low IRS‐1 protein expression in fat cells as well as the potential molecular mechanisms related to the adipose tissue. Twenty‐five non‐obese individuals with low or normal IRS‐1 expression in subcutaneous abdominal fat cells were extensively characterized and the results compared with 71 carefully matched subjects with or without a known genetic predisposition for type 2 diabetes. In contrast to the commonlyused risk marker, knownheredity for diabetes, low cellular IRS‐1 identified individuals who were markedly insulin resistant, had high proinsulin and insulin levels, and exhibited evidence of early atherosclerosis measured as increased intima media thickness in the carotid artery bulb. Circulating levels of adiponectin were also significantly reduced. Gene analyses of fat cells in a parallel study showed attenuated expression of several genes related to fat cell differentiation (adiponectin, aP2, PPARγ, and lipoprotein lipase) in the group of individuals characterized by a low IRS‐1 expression and insulin resistance. A low IRS‐1 expression in fat cells is a marker of insulin resistance and risk for type 2 diabetes and is associated with evidence of early vascular complications. Impaired adipocyte differentiation, including low gene expression and circulating levels of adiponectin, can provide a link between the cellular marker and the in vivo phenotype.—Jansson, P.‐A., Pellmé, F., Hammarstedt, A., Sandqvist, M., Brekke, H., Caidahl, K., Forsberg, M., Volkmann, R., Carvalho, E., Funahashi, T., Matsuzawa, Y., Wiklund, O., Yang, X., Taskinen, M.‐R., Smith, U. A novel cellular marker of insulin resistance and early atherosclerosis in humans is related to impaired fat cell differentiation and low adiponectin. FASEB J. 17, 1434–1440 (2003)

Insulin Signaling and Action in Fat Cells: Associations with Insulin Resistance and Type 2 Diabetes

ABSTRACT: Adipose tissue only accounts for a relatively small proportion (<10%) of the peripheral glucose utilization in response to insulin. However, the fat cells may still play an important role in insulin resistance and Syndrome X through, for instance, its endocrine functions (production of leptin, TNFα, PAI‐1, etc.) and involvement in lipid metabolism (FFA release and hydrolysis of triglycerides). The fat cells are also highly sensitive to insulin and may thus be used to elucidate molecular mechanisms for insulin resistance in man. Examinations of the intracellular signaling mechanisms for insulin in fat cells from individuals with Type 2 diabetes revealed markedly lower insulin‐stimulated PI3‐kinase activity. This was due to a pronounced reduction in the cellular expression of the docking protein, IRS 1, whereas expression of IRS 2 was normal. However, IRS 2‐associated PI3‐kinase activity was only approximately one‐third of that found to be associated with IRS 1 in normal cells.

mTOR inhibition with rapamycin causes impaired insulin signalling and glucose uptake in human subcutaneous and omental adipocytes

Rapamycin is an immunosuppressive agent used after organ transplantation, but its molecular effects on glucose metabolism needs further evaluation. We explored rapamycin effects on glucose uptake and insulin signalling proteins in adipocytes obtained via subcutaneous (n=62) and omental (n=10) fat biopsies in human donors. At therapeutic concentration (0.01 μM) rapamycin reduced basal and insulin-stimulated glucose uptake by 20-30%, after short-term (15 min) or long-term (20 h) culture of subcutaneous (n=23 and n=10) and omental adipocytes (n=6 and n=7). Rapamycin reduced PKB Ser473 and AS160 Thr642 phosphorylation, and IRS2 protein levels in subcutaneous adipocytes. Additionally, it reduced mTOR-raptor, mTOR-rictor and mTOR-Sin1 interactions, suggesting decreased mTORC1 and mTORC2 formation. Rapamycin also reduced IR Tyr1146 and IRS1 Ser307/Ser616/Ser636 phosphorylation, whereas no effects were observed on the insulin stimulated IRS1-Tyr and TSC2 Thr1462 phosphorylation. This is the first study to show that rapamycin reduces glucose uptake in human adipocytes through impaired insulin signalling and this may contribute to the development of insulin resistance associated with rapamycin therapy.

Impaired glucose transport and protein kinase B activation by insulin, but not okadaic acid, in adipocytes from subjects with Type II diabetes mellitus

Aims/hypothesis. To study the effects of insulin and okadaic acid, a serine/threonine phosphatase inhibitor which does not increase PI3-kinase activity, on the rate of glucose transport and protein kinase B activation in adipocytes from healthy subjects and subjects with Type II (non-insulin-dependent) diabetes mellitus. Methods. Adipocytes were incubated with or without insulin or okadaic acid or both and glucose transport, protein kinase B activity, phosphorylation and protein expression measured. Results. Insulin and okadaic acid alone increased glucose uptake to a similar degree in adipocytes from healthy subjects and, when combined, exerted a partial additive effect. The effect of insulin was reduced by about 60 % in adipocytes from Type II diabetic patients, whereas the effect of okadaic acid was essentially unchanged and no further increase was seen when okadaic acid and insulin were combined. Okadaic acid increased protein kinase B activity to a greater extent (two to threefold) than insulin but only slightly increased the serine phosphorylation of protein kinase B. Adipocytes from Type II diabetic subjects exhibited both an impaired sensitivity as well as a reduced total serine phosphorylation and activation of protein kinase B in response to insulin but protein kinase B activity in response to okadaic acid was intact. Conclusion/interpretation. These results show that the ability of insulin to increase glucose transport and activate protein kinase B is reduced in fat cells from Type II diabetic subjects. Protein kinase B can, however, be activated by agents like okadaic acid which bypass the upstream defects in the insulin signalling pathway in Type II diabetic cells and, thus, increase glucose uptake. [Diabetologia (1999) 42: 819–825]

Insulin resistance in fat cells from obese Zucker rats--evidence for an impaired activation and translocation of protein kinase B and glucose transporter 4.

The effect of insulin on glucose transport, glucose transporter 4 (Glut4) translocation, and intracellular signaling were measured in fat cells from lean and obese Zucker rats of different ages. Insulin-stimulated glucose transport was markedly reduced in adipocytes from old and obese animals. The protein content of Glut4 and insulin receptor substrates (IRS) 1 and 2 were also reduced while other proteins, including the p85 subunit of PI3-kinase, Shc and the MAP kinases (ERK1 and 2) were essentially unchanged. There was a marked impairment in the insulin stimulated tyrosine phosphorylation of IRS-1 and 2 as well as activation of PI3-kinase and PKB in cells from old and obese animals. Furthermore, insulin-stimulated translocation of both Glut4 and PKB to the plasma membrane was virtually abolished. The phosphotyrosine phosphatase inhibitor, vanadate, increased the insulin- stimulated upstream signaling including PI3-kinase and PKB activities as well as rate of glucose transport. Thus, the insulin resistance in cells from old and obese Zucker rats can be accounted for by an impaired translocation process, due to signaling defects leading to a reduced activation of PI3-kinase and PKB, as well as an attenuated Glut4 protein content.

Cellular cross-talk between epicardial adipose tissue and myocardium in relation to the pathogenesis of cardiovascular disease

Epicardial and perivascular fat depot size is considered an index of cardiac and visceral obesity. The functional and anatomic proximity of epicardial adipose tissue (EAT) to myocardium has drawn increasing attention in recent years among researchers attempting to elucidate its putative role as an endocrine organ. This includes the role of EAT as a lipid storing depot and as an inflammatory tissue secreting cytokines and chemokines under pathogenic conditions such as cardiovascular diseases. In this review, we discuss the current state of knowledge regarding the potential EAT mediators of inflammation and the paracrine cross-talk between EAT and the underlying myocardium. We also highlight the most recent findings on the causes and correlates of myocardial steatosis/cardiac lipotoxicity and its association with cardiac dysfunction.

Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from Type II diabetic subjects.

Aims/hypothesis. To examine protein kinase B/Akt distribution and phosphorylation in response to insulin in different subcellular fractions of human fat cells from healthy subjects and subjects with Type II (non-insulin-dependent) diabetes mellitus. Methods. We prepared subcellular fractions of plasma membranes (PM), low density microsomes and cytosol and examined gene and protein expression as well as serine and threonine phosphorylation in response to insulin. Results. Protein kinase B/Akt mRNA as well as total protein kinase B/Akt protein in whole-cell lysate and cytosol were similar in both groups. Insulin increased protein kinase B/Akt translocation to the the plasma membrane about twofold [(p < 0.03) in non-diabetic cells but this effect was impaired in diabetic cells (∼ 30 %; p > 0.1)]. In both groups, protein kinase B/Akt threonine phosphorylation considerably increased in low density microsomes and cytosol whereas serine phosphorylation was predominant in the plasma membrane. Phosphatidylinositol-dependent kinase 1, which partially activates and phosphorylates protein kinase B/Akt on the specific threonine site, was predominant in cytosol but it was also recovered in low density microsomes. Serine phosphorylation in response to insulin was considerably reduced (50–70 %; p < 0.05) in diabetic cells but threonine phosphorylation was less reduced (∼ 20 %). Wortmannin inhibited these effects of insulin supporting a role for PI3-kinase activation. Conclusion/interpretation. Insulin stimulates a differential subcellular pattern of phosphorylation of protein kinase B/Akt. Furthermore, insulin-stimulated translocation of protein kinase B/Akt to the plasma membrane, where serine phosphorylation and full activation occurs, is impaired in Type II diabetes. Threonine phosphorylation was much less reduced. This discrepancy may be related to differential activation of phosphatidylinositol 3-kinase in the different subcellular compartments and phosphatidylinositol-dependent kinase 1 having high affinity for phosphatidylinositol phosphate 3. [Diabetologia (2000) 43: 1107–1115]

Improved Survival, Vascular Differentiation and Wound Healing Potential of Stem Cells Co-Cultured with Endothelial Cells

In this study, we developed a methodology to improve the survival, vascular differentiation and regenerative potential of umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34+ cells), by co-culturing the stem cells in a 3D fibrin gel with CD34+-derived endothelial cells (ECs). ECs differentiated from CD34+ cells appear to have superior angiogenic properties to fully differentiated ECs, such as human umbilical vein endothelial cells (HUVECs). Our results indicate that the pro-survival effect of CD34+-derived ECs on CD34+ cells is mediated, at least in part, by bioactive factors released from ECs. This effect likely involves the secretion of novel cytokines, including interleukin-17 (IL-17) and interleukin-10 (IL-10), and the activation of the ERK 1/2 pathway in CD34+ cells. We also show that the endothelial differentiation of CD34+ cells in co-culture with CD34+-derived ECs is mediated by a combination of soluble and insoluble factors. The regenerative potential of this co-culture system was demonstrated in a chronic wound diabetic animal model. The co-transplantation of CD34+ cells with CD34+-derived ECs improved the wound healing relatively to controls, by decreasing the inflammatory reaction and increasing the neovascularization of the wound.