Walid Kuri-Harcuch

Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O

SummaryCultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.

Thyroid hormone stimulates adipocyte differentiation of 3T3 cells.

Triiodothyronine added at 0.1 nM to 3T3-F442A cells cultured in adipogenic medium having endogenous hormone concentrations similar to those of hypothyroid serum stimulated adipose conversion; activities of both lipogenic enzymes, glycerophosphate dehydrogenase and malic enzyme, increased with hormone treatment. The number of adipocytes was also augmented by L-T3 addition but the number of fat cell clusters remained the same as compared to non-treated cultures, suggesting that thyroid hormone increased the number of adipocytes probably through stimulating selective multiplication of precursor adipose cells. Hormone addition to cells cultured with non-adipogenic medium did not promote conversion showing that L-T3 is not an adipogenic factor by itself. Triiodothyronine added at concentrations similar to those found in hyperthyroidism, from 10 nM up to 10 µM, also increased the proportion of adipocytes without changing the number of fat cell clusters, but they decreased the activity of both lipogenic enzymes and lipid accumulation in mature adipocytes. It can be concluded that during 3T3-F442A differentiation into adipocytes L-T3 increases the number of differentiated adipocytes and, at low concentrations, also enhances lipogenic enzyme activities, whereas at the hyperthyroid hormone levels these enzyme activities are significantly reduced, remaining at levels similar to those of cells cultured with hypothyroid medium. This cloned cell line seems to be a useful model to study thyroid hormone action at both molecular and cellular level.

Controlled clinical study of skin donor sites and deep partial-thickness burns treated with cultured epidermal allografts.

Two clinical studies in donor sites and deep partial-thickness burns treated with banked cultured human epidermal allografts are described. Ten burn patients were subjected to donor split-thickness skin harvesting. The study was controlled, side-by-side comparative, blind, and randomized. Banked cultured epidermal allografts promoted a faster reepithelialization of the wounds; they epithelialized in an average of 6.9 days, whereas controls healed in an average of 11.1 days, giving a reduction of 37.8 percent in time to heal (p < 0.005). Allografted sites were less erythematous as compared with controls (p < 0.01), with more tendency to normopigmentation. In the deep partial-thickness burns study, 10 patients with 18 burned wounds were treated. Wounds treated with cultured allografts showed complete reepithelialization in about 3 to 6 days. The two clinical studies showed that banked cultured epidermal allograft promotes a significantly faster epithelialization of donor sites and deep partial-thickness wounds. These results support the idea that cultured allografts should be used routinely to improve treatment of burn patients and reduce their therapy time.

Growth factors and extracellular matrix proteins during wound healing promoted with frozen cultured sheets of human epidermal keratinocytes

Abstract. In a murine model of full-thickness wounds, healing is stimulated by the application of human frozen cultured epidermal sheets. With immunofluorescence techniques, we studied, during this process, the spatial and temporal pattern of expression of: transforming growth factor-α (TGF-α); transforming growth factor-β (TGF-β) isoforms 1, 2, and 3; platelet-derived growth factor (PDGF); and the extracellular matrix proteins fibronectin, collagen IV, and tenascin. The growth factors, with the exception of PDGF, were found to be located in the frozen cultured sheet of keratinocytes before and after its application to the wound, whereas collagen IV and tenascin were deposited in the connective tissue under the frozen cultures. None of these factors were detected in control wound beds. Monoclonal antibodies against collagen IV and tenascin showed that both were of murine origin. We propose that the frozen cultures of human keratinocytes promote faster reepithelialization through the release of growth factors such as TGF-α which directly enhance migration and proliferation of murine keratinocytes, and through the stimulation of murine subepithelial cells, by TGF-β, to secrete basement membrane proteins such as collagen IV, laminin, and tenascin, which provide a provisional substrate that improves migration of the murine epidermal cells.

Commitment of 3T3-F442A cells to adipocyte differentiation takes place during the first 24–36 h after adipogenic stimulation: TNF-α inhibits commitment

We studied the commitment of 3T3-F442A cells during stimulation with adipogenic serum or growth hormone. Confluent 3T3-F442A preadipocytes were incubated with adipogenic medium for increasing times; the number of adipose clusters, GPDH activity, and lipid accumulation were evaluated. Results show that cell commitment took place during the first 24-36 h after stimulation under adipogenic conditions. Then, cultures underwent a 2-fold increase in total cell number through selective multiplication of committed cells, followed by a dramatic decrease in colony-forming ability and 300- to 1000-fold raise in GPDH activity. Cell commitment was not modulated by insulin, but this hormone stimulated clonal expansion of committed cells and lipogenesis. Commitment was inhibited by TNF-alpha at concentrations as low as 5 ng/ml, and by retinoic acid. The results show that TNF-alpha inhibits adipose conversion at two different levels: at concentrations as low as 5 ng/ml, it blocks commitment, and at concentrations of 100 ng/ml or higher the cytokine seems to block mitotic expansion and other steps of differentiation after cell commitment. The identification of a specific time for cell commitment would allow the study of the early genes that might regulate cell reprogramming into adipocytes.

Combined use of allograft and autograft epidermal cultures in therapy of burns.

&NA; Cultivation of human epidermal keratinocytes made possible the use of cultured autografts as part of the therapy of extensively burned patients. On the basis of our early results using banked cultured allografts and autografts, we developed an integral and combined burn therapy comprising banked cultured allografts for rapid healing of skin donor sites and deep partial‐thickness burns, conventional split‐thickness skin autografting, and when needed, cultured autografts for full‐thickness burns. We compared hospital stay in 32 burn patients treated with the combined therapy and in 39 who were not treated with cultured epidermis. Three groups of patients were defined: 15 to 29 percent (n = 12), 30 to 49 percent (n = 10), and more than 49 percent (n = 10) burned body surface area. We found a 20 to 29 percent decrease in hospital stay in patients with up to 49 percent burned body surface area and a 46 percent reduction in patients suffering more extensive burns. Survival rate of extensively burned patients also was increased. We took advantage of the availability of banked cultured allografts for ambulatory treatment, without hospitalization, of pediatric patients with 5 to 20 percent burned body surface area. We show for the first time the use and benefits of this combined therapy.

Glucocorticoid Paradoxically Recruits Adipose Progenitors and Impairs Lipid Homeostasis and Glucose Transport in Mature Adipocytes

Chronic treatment with glucocorticoids increases the mass of adipose tissue and promotes metabolic syndrome. However little is known about the molecular effects of dexamethasone on adipose biology. Here, we demonstrated that dexamethasone induces progenitor cells to undergo adipogenesis. In the adipogenic pathway, at least two cell types are found: cells with the susceptibility to undergo staurosporine-induced adipose conversion and cells that require both staurosporine and dexamethasone to undergo adipogenesis. Dexamethasone increased and accelerated the expression of main adipogenic genes such as pparg2, cebpa and srebf1c. Also, dexamethasone altered the phosphorylation pattern of C/EBPβ, which is an important transcription factor during adipogenesis. Dexamethasone also had effect on mature adipocytes mature adipocytes causing the downregulation of some lipogenic genes, promoted a lipolysis state, and decreased the uptake of glucose. These paradoxical effects appear to explain the complexity of the action of glucocorticoids, which involves the hyperplasia of adipose cells and insulin resistance.

Frozen cultured sheets of human epidermal keratinocytes enhance healing of full-thickness wounds in mice

Abstract Sheets of cultured allogeneic human keratinocytes have been used for the treatment of burns and chronic leg ulcers but there has been no animal assay for the therapeutic action of these cultures. In order to analyze the effects of frozen cultures of human keratinocytes on wound healing, we have developed such an assay based on the rate of repair of full-thickness skin wounds in immunocompetent NMR1 mice. Reepithelialization of the control wounds, originating from the murine epithelium at the edge of the wound, occurred at a constant rate of advance of 150 µm/day. When frozen cultured human epidermal sheets were thawed at room temperature for 5–10 min and applied to the surface of the wound, the murine epithelium advanced at 267 µm/day. Most wounds treated with frozen cultures completely healed after 10 days, whereas most control wounds required 16 days. The accelerated reepithelialization did not depend on the presence of proliferative human keratinocytes in the frozen cultures. The cultures also promoted early formation of granulation tissue and laminin deposition over the surface of the wound bed. This simple assay should permit quantitative analysis of the effects on healing exerted not only by cultured cells, but also by proteins and small molecules.

Fibromodulin gene is expressed in human epidermal keratinocytes in culture and in human epidermis in vivo

Fibromodulin is a small leucine-rich proteoglycan that has a central role in the maintenance of collagen fibrils structure, and in regulation of TGF-beta biological activity. Although, it is mainly found in cartilage and tendon, little is known regarding the expression of the fibromodulin gene in other cell types. By RT-PCR, real time PCR and immunohistochemistry, we describe the expression of the fibromodulin gene and the presence of the protein in human epidermal keratinocytes (HEK), both in culture and in normal human epidermis. Our results show, for the first time, that fibromodulin gene is constantly expressed in HEK during culture time. Immunostaining showed that fibromodulin is located intracytoplasmically in basal and stratified keratinocytes of the growing colonies, confluent cultures, and epidermis in vivo. The expression and intracellular localization of fibromodulin in HEK is a new finding and opens new possible biological roles for the SLRP family.

Staurosporine rapidly commits 3T3-F442A cells to the formation of adipocytes by activation of GSK-3β and mobilization of calcium

Pre‐adipose 3T3‐F442A cells exposed to fetal bovine serum or human growth hormone (adipogenic medium) become irreversibly committed to differentiation into adipocytes within 24–36 h. We show now that the action of the serine‐threonine kinase inhibitor staurosporine is much more rapid since its addition in non‐adipogenic medium resulted in commitment to adipocyte differentiation within 4–6 h. During this period, glycogen synthase kinase 3β was activated. Commitment depended on an increase in the intracellular calcium concentration that was modulated in part by a T‐type calcium channel since mibefradil, amiloride, and NiCl2, which are selective blockers of the T‐type channels, partially inhibited adipose differentiation. Studies of the inhibitory action of retinoic acid showed that a period of time after exposure to St was required in order to stabilize the commitment to adipose differentiation. It was concluded that the commitment of the cells consists of two stages. Commitment is promoted during the first one, and during the second there is a stabilization which still can be destabilized by the addition of retinoic acid or other drugs. The commitment becomes stable after 40 h of staurosporine treatment, and can no longer be prevented by retinoic acid. The identification of these two stages of commitment makes it possible to analyze in further detail early molecular events of the process and the nature of any other participating genes. J. Cell. Biochem. 105: 147–157, 2008.