Peter Gál

Human Galectins Induce Conversion of Dermal Fibroblasts into Myofibroblasts and Production of Extracellular Matrix: Potential Application in Tissue Engineering and Wound Repair

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multifunctionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-β1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum.

Soy and Breast Cancer: Focus on Angiogenesis

Epidemiological studies have revealed that high consumption of soy products is associated with low incidences of hormone-dependent cancers, including breast and prostate cancer. Soybeans contain large amounts of isoflavones, such as the genistein and daidzain. Previously, it has been demonstrated that genistein, one of the predominant soy isoflavones, can inhibit several steps involved in carcinogenesis. It is suggested that genistein possesses pleiotropic molecular mechanisms of action including inhibition of tyrosine kinases, DNA topoisomerase II, 5α-reductase, galectin-induced G2/M arrest, protein histidine kinase, and cyclin-dependent kinases, modulation of different signaling pathways associated with the growth of cancer cells (e.g., NF-κB, Akt, MAPK), etc. Moreover, genistein is also a potent inhibitor of angiogenesis. Uncontrolled angiogenesis is considered as a key step in cancer growth, invasion, and metastasis. Genistein was found to inhibit angiogenesis through regulation of multiple pathways, such as regulation of VEGF, MMPs, EGFR expressions and NF-κB, PI3-K/Akt, ERK1/2 signaling pathways, thereby causing strong antiangiogenic effects. This review focuses on the antiangiogenic properties of soy isoflavonoids and examines their possible underlying mechanisms.

Open Wound Healing In Vivo: Monitoring Binding and Presence of Adhesion/Growth-Regulatory Galectins in Rat Skin during the Course of Complete Re-Epithelialization

Galectins are a family of carbohydrate-binding proteins that modulate inflammation and immunity. This functional versatility prompted us to perform a histochemical study of their occurrence during wound healing using rat skin as an in vivo model. Wound healing is a dynamic process that exhibits three basic phases: inflammation, proliferation, and maturation. In this study antibodies against keratins-10 and -14, wide-spectrum cytokeratin, vimentin, and fibronectin, and non-cross-reactive antibodies to galectins-1, -2, and -3 were applied to frozen sections of skin specimens two days (inflammatory phase), seven days (proliferation phase), and twenty-one days (maturation phase) after wounding. The presence of binding sites for galectins-1, -2, -3, and -7 as a measure for assessing changes in reactivity was determined using labeled proteins as probes. Our study detected a series of alterations in galectin parameters during the different phases of wound healing. Presence of galectin-1, for example, increased during the early phase of healing, whereas galectin-3 rapidly decreased in newly formed granulation tissue. In addition, nuclear reactivity of epidermal cells for galectin-2 occurred seven days post-trauma. The dynamic regulation of galectins during re-epithelialization intimates a role of these proteins in skin wound healing, most notably for galectin-1 increasing during the early phases and galectin-3 then slightly increasing during later phases of healing. Such changes may identify a potential target for the development of novel drugs to aid in wound repair and patients’ care.

The Effect of Equal Daily Dose Achieved by Different Power Densities of Low-Level Laser Therapy at 635 and 670 nm on Wound Tensile Strength in Rats: A Short Report

OBJECTIVE The aim of our study was to compare the effects of different power densities of LLLT at 635 and 670 nm achieving a daily dose of 5 J/cm(2) on wound tensile strength (TS) in rats. BACKGROUND DATA Optimal parameters of low-level laser therapy (LLLT) are still unknown. MATERIALS AND METHODS Under general anesthesia, one full-thickness skin incision was performed on the back of each rat (n = 40) and immediately closed using an intradermal running suture. Rats were separated into five groups depending on treatment parameters: (1) sham irradiated control group (SIC); (2) 635 nm laser-treated group at 4 mW/cm(2) (L-635/4); (3) 635 nm laser-treated group at 15 mW/cm(2) (L-635/15); (4) 670 nm laser-treated group at 4 mW/cm(2) (L-670/4); and (5) 670 nm laser-treated group at 15 mW/cm(2) (L-670/15). The total daily dose was 5 J/cm(2). Seven days after surgery each wound was removed for wound TS measurement. RESULTS The lowest wound TS results were measured in the SIC rats (10.5 +/- 2.8 g/mm(2)). Higher wound TS results were measured in group L-670/15 (11.5 +/- 2.5 g/mm(2)) and group L-635/4 (11.7 +/- 4.3 g/mm(2)) rats, while significantly higher results were found in group L-670/4 (15.8 +/- 4.4 g/mm(2)) and group L-635/15 (15.9 +/- 4.8 g/mm(2)). The differences were significant between certain groups (p < 0.01: SIC vs. L-635/15, SIC vs. L-670/4; p < 0.05: L-635/4 vs. L-635/15, L-635/4 vs. L-670/4, L-635/15 vs. L-670/15, L-670/4 vs. L-670/15). CONCLUSION Both red lasers significantly increased wound TS at selected parameters. Whereas the 635 nm laser significantly improved wound healing by using the higher power density, the 670 nm laser improved healing using a lower power density.

Effect of Atropa belladonna L. on skin wound healing: biomechanical and histological study in rats and in vitro study in keratinocytes, 3T3 fibroblasts, and human umbilical vein endothelial cells.

The effect of Atropa belladonna L. (AB) aqueous extract on skin wound healing was studied in male Sprague–Dawley rats subjected to two parallel full‐thickness skin incisions on the back. Specimens for histological evaluation were collected on days 2 and 5 whereas for biomechanical testing, they were collected on day 5. In the in vitro study, a different concentration of AB extract was used to test the differentiation of keratinocytes using a panel of selected antibodies, proliferation, and cell survival of 3T3 fibroblasts and human umbilical vein endothelial cells using the MTT‐assay. Results of the in vivo experiments showed in AB‐treated wounds a shortened process of inflammation and accelerated collagen formation, as well as significantly increased wound stiffness as compared with control tissues. The in vitro examination showed that control keratinocytes were cytokeratin 19 free, while samples exposed to the highest AB extract concentration expressed CK19. Moreover, all concentrations were stimulatory to human umbilical vein endothelial cell proliferation. In addition, only the AB extract at the lowest tested concentration increased fibroblast growth, but higher concentrations decreased cell survival. In conclusion, our results indicate that the AB water extract positively affects early phases of skin wound healing in rats. However, the in vitro results on the inverse relation between the concentration of the AB extract and its effects on cell proliferation may be important for future research.

Chemokines as Possible Targets in Modulation of the Secondary Damage After Acute Spinal Cord Injury: A Review

In spite of many promising experimental studies, an effective treatment dramatically eliminating the secondary damage after spinal cord injury (SCI) is still missing. Since clinical data on the therapeutical effect after methylprednisolone treatment are not conclusive, new therapeutical modalities targeting specific components of secondary spinal cord damage needs to be developed. It is known that immune cells are recruited to injury sites by chemokines, which are small, structurally similar proteins released locally at the site of inflammation. Hence, this review was aimed to summarize possible roles of chemokines in the inflammation following SCI as well as to identify possible new therapeutical targets which can potentially be effective in ameliorating individual components of this inflammatory response. Data concerning inflammation reduction together with techniques improving axonal growth, cell replacement and remyelinization, may be crucial to move a small step forward in an attempt to make paraplegic and quadriplegic patients to walk.

ER-α agonist induces conversion of fibroblasts into myofibroblasts, while ER-β agonist increases ECM production and wound tensile strength of healing skin wounds in ovariectomised rats.

Abstract:  Oestrogen deprivation is one of the major factors responsible for many age‐related processes, including poor wound healing in women. Previously, it has been shown that oestrogens have a modulatory effect in different wound‐healing models. Therefore, in this study, the effect of selective oestrogen receptor (ER) agonists (PPT – ER‐α agonist, DPN – ER‐β agonist) on excisional and incisional wound‐healing models was compared in ovariectomised rats in vivo as well as on human dermal fibroblasts (HDF) and human umbilical endothelial cells (HUVEC) in vitro. In the in vivo study, 4 months after either ovariectomy or sham ovariectomy, Sprague‐Dawley rats were randomly divided into four groups and subjected to two incisional and excisional wounds: (i) control – sham operated, vehicle‐treated; (ii) ovariectomised, vehicle‐treated; (iii) ovariectomised, PPT treated; (iv) ovariectomised, DPN treated. In the in vitro study, HDFs and HUVECs were used. After treatment with ER agonists, cells were processed for immunocytochemistry and gelatin zymography. Our study shows that stimulation of ER‐α leads to the differentiation of fibroblasts into myofibroblasts both in vivo and in vitro. On the other hand, the formation of extracellular matrix was more prominent, and wound tensile strength (TS) was increased when ER‐β was stimulated. In contrast, stimulation of ER‐α led to a more prominent increase in the expression of MMP‐2 and decrease in wound TS. New information is presented in this investigation concerning oestrogen replacement therapy (ERT) in different wound‐healing models. This study demonstrates that the ERT should be both wound and receptor‐type specific.

In Vivo Monitoring the Changes of Interstitial pH and FAD/NADH Ratio by Fluorescence Spectroscopy in Healing Skin Wounds

Abstract The aim of our study was to evaluate the changes of interstitial pH and flavin adenine dinucleotide (FAD)/reduced nicotinamide adenine dinucleotide (NADH) ratio in healing skin wounds using fluorescence spectroscopy in Sprague Dawley rats. In the experiment, excisional and incisional models of wound healing were used. The florescein as the pH-sensitive probe using excitation spectra (λEm = 535 nm) was used for the measurement of pH changes, and synchronous fluorescence spectra (Δλ = 60 nm) for the monitoring of FAD/NADH ratio changes were measured from the surfaces of healing wounds. Increase of interstitial pH and FAD/NADH ratio was recorded during the time interval from the 15th to the 65th minute after surgery. The decrease of pH between the 48th and the 72nd hour after surgery as well as the increase of FAD/NADH ratio between the 72nd and the 96th hour of wound healing were recorded. The results indicate that the use of fluorescence spectroscopy may be considered as a valuable tool for noninvasive in vivo monitoring of selected redox parameters in the early phases of wound healing.

Low-Level Laser Therapy with 810 nm Wavelength Improves Skin Wound Healing in Rats with Streptozotocin-Induced Diabetes

OBJECTIVE The aim of present study was to evaluate whether low-level laser therapy (LLLT) can reverse the impaired wound healing process in diabetic rats. BACKGROUND DATA Impaired wound healing in diabetic patients represents a major health problem. Recent studies have indicated that LLLT may improve wound healing in diabetic rats, but the optimal treatment parameters are still unknown. MATERIALS AND METHODS Male Sprague-Dawley rats (n=21) were randomly divided into three groups: a healthy control group, a diabetic sham-treated group, and a diabetic LLLT-treated group. Diabetes mellitus was then induced by streptozotocin administration to the two diabetic groups. One 4 cm long full thickness skin incision and one full thickness circular excision (diameter=4 mm) were performed on the back of each rat. An infrared 810 nm laser with an output of 30 mW, a power density of 30 mW/cm(2), and a spot size of 1 cm(2) was used to irradiate each wound for 30 sec (daily dose of 0.9 J/cm(2)/wound/day). RESULTS In diabetic rats, the histology of LLLT-treated excisions revealed a similar healing response to that in nondiabetic controls, with significantly more mature granulation tissue than in the sham-treated diabetic control group. LLLT reduced the loss of tensile strength, and increased the incision wound stiffness significantly compared with sham-irradiated rats, but this did not achieve the same level as in the nondiabetic controls. CONCLUSIONS Our study demonstrates that infrared LLLT can improve wound healing in diabetic rats. Nevertheless, further research needs to be performed to evaluate the exact underlying mechanism and to further optimize LLLT parameters for clinical use.

Agrimonia eupatoria L. and Cynara cardunculus L. Water Infusions: Phenolic Profile and Comparison of Antioxidant Activities

Reactive oxygen species (ROS) are highly considered in the ethiopathogenesis of different pathological conditions because they may cause significant damage to cells and tissues. In this paper, we focused on potential antioxidant properties of two medical plants such as the Agrimonia eupatoria L. and Cynara cardunculus L. Both plants have previously been studied for their pharmacological activities, especially as hepatoprotective and hypoglycemic activities. It has been suggested, that their effects are related to the antioxidant properties of polyphenols, which are dominant compounds of the plants’ extracts. In the present study HPLC-MS analysis of water infusion was performed allowing the identification of several phenolic constituents. Furthermore, antioxidant effects of the two extracts were compared showing higher effects for agrimony extract compared to artichoke. Thus, agrimony was selected for the in vivo study using the skin flap viability model. In conclusion, our results provide evidence that the A. eupatoria extract may be a valuable source of polyphenols to be studied for the future development of supplements useful in the prevention of diseases linked to oxidative stress.