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In this paper we address the linking of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF-2) to intracellular signaling molecules in oligodendrocyte progenitors. It is demonstrated that both growth factors activate downstream targets similar to those shown for protein kinase C (PKC) activation. Yet, neither the arrest of terminal oligodendrocyte differentiation nor the proliferation induced by PDGF or FGF-5 can be antagonized by inhibition of PKC. Rather, p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, and pp70 S6 kinase were found to be necessary for the mitogenic activity of PDGF and FGF-2. Paradoxically, these kinases were also necessary for the onset of oligodendrocyte differentiation in control cells. In addition, cAMP-dependent kinase A (PKA) activation inhibited the mitogenic response of oligodendrocyte progenitors to FGF-P. Taken together, the molecular mechanism that controls oligodendrocyte lineage progression is operated by at least two signal pathways, which interfere either with proliferation and/or differentiation of oligodendrocyte progenitors.

The factors that regulate the ontogeny and differentiation of C1 adrenergic neurons located in the rostral ventrolateral medulla (RVLM) are completely unknown. In the present study, we have investigated the effects of a number of neurotrophic factors on the survival of E18-19 rat C1 adrenergic neurons in culture. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to study the expression of tyrosine hydroxylase (TH), an enzyme present in all catecholaminergic neurons, and of phenylethanolamine N-methyltransferase (PNMT), the final enzyme in the synthesis of adrenalin, as markers for the C1 RVLM neurons. Our results show that GDNF, CNTF BDNF, NT-3 and NT-4/5 increase the number of TH-immunoreactive neurons surviving in vitro. The effects of NGF, TGF beta and bFGF were not significant. The E18-19 CI neurons appeared to loose their ability to express PNMT in culture as examined with immunocytochemistry and RT-PCR, and none of the tested neurotrophic factors was able to sustain or induce this expression. Our results indicate that the adrenergic phenotype of C1 neurons, or the survival of these neurons, is determined by environmental factors other than the neurotrophic factors examined in this study. (C) 1999 Elsevier Science B.V. All rights reserved.

Fibrin or a fibrinous exudate can facilitate angiogenesis in many pathological conditions. In vitro, the outgrowth of capillary-like structures in fibrin can be mimicked by exposing human microvascular endothelial cells (hMVECs) to an angiogenic growth factor and tumor necrosis factor (TNF)-alpha. Urokinase-type plasminogen activator (u-PA) and plasmin activities are required for this angiogenic process. This study focuses on the role and localization of the u-PA receptor (u-PAR) in newly formed microvascular structures. The u-PAR-blocking monoclonal antibody (MAb) H-2 completely inhibited the formation of capillary-like tubular structures induced by exposure of hMVECs to basic fibroblast growth factor and TNF-alpha. This was accompanied by a several-fold increase in u-PA accumulation in the conditioned medium. The effect of MAb H-2 was not caused by blocking cellular activation by u-PA/u-PAR interaction, as the aminoterminal fragment (ATF) of u-PA, which also activates u-PAR, prevented tube formation. In addition, the inhibition by MAb H-2 was not due to an effect of the antibody on u-PAR-vitronectin binding. These data show that inhibition of tube formation can be caused not only by inhibition of u-PA or plasmin activities but also by unavailability of the u-PAR for cell-bound proteolysis. Immunohistochemical analysis showed that in in vitro angiogenesis u-PAR and u-PA were localized on the invading, tube-forming hMVECs and not on the endothelial cells that are located on top of the fibrin matrix. u-PAR and u-PA were also prominently expressed on endothelial cells of neovessels present in an atherosclerotic plaque. These data may give more insight into the role of u-PAR in repair-associated angiogenesis.

The role of epidermal keratinocytes in the early phases of normal unimpaired wound healing has been studied extensively. However, little is known about the cell biological processes in the epidermis and the basal membrane zone during the later phases of dermal matrix formation and remodelling of the scar tissue, This study investigated epidermal growth and differentiation and maturation of the basal membrane zone. Biopsies mere taken from (clinically) hypertrophic and non-hypertrophic scars at 3 and 12 months after a breast-reduction operation, Tissues were analysed using immunohistochemical techniques. The data showed that epidermal abnormalities with respect to differentiation persist up to 3 months, as witnessed by the expression of cytokeratin 16, Remarkably, hypertrophic scars that remained hypertrophic throughout the period of analysis (up to 12 months) showed significantly more cytokeratin 16 expression at 3 months, when compared tither with normal scars or with hypertrophic scars that became normal after 12 months, Staining for Ki-67 antigen, a marker for cell proliferation, revealed an increase in basal keratinocyte proliferation rate in 3-month-old hypertrophic scars compared with non-hypertrophic scars. After 12 months, this difference had disappeared completely and the number of cycling basal cells had returned to normal values, Three-month-old hypertrophic scars showed more acanthosis than non-hypertrophic scars of the same age, irrespective of whether they remained hypertrophic or became normal scars. After 12 months, this difference was no longer present, Staining for various heparan sulphate proteoglycan epitopes revealed that restoration of the basal membrane was incomplete at 3 months, but was complete at 12 months with respect to this component, No differences in the expression of several components of the basal membrane zone (heparan sulphate proteoglycan, laminin, tenascin) were noted between hypertrophic and non-hypertrophic scars, These data show that in the early phase of hypertrophic scarring, epidermal abnormalities are found compared with normal wound healing, In addition, early (3 months) epidermal abnormalities are associated with the clinical outcome at 12 months, These findings raise the possibility that the epidermal compartment is involved in the pathogenic process, (C) 1998 John Wiley & Sons, Ltd.

The role of epidermal keratinocytes in the early phases of normal unimpaired wound healing has been studied extensively. However, little is known about the cell biological processes in the epidermis and the basal membrane zone during the later phases of dermal matrix formation and remodelling of the scar tissue, This study investigated epidermal growth and differentiation and maturation of the basal membrane zone. Biopsies mere taken from (clinically) hypertrophic and non-hypertrophic scars at 3 and 12 months after a breast-reduction operation, Tissues were analysed using immunohistochemical techniques. The data showed that epidermal abnormalities with respect to differentiation persist up to 3 months, as witnessed by the expression of cytokeratin 16, Remarkably, hypertrophic scars that remained hypertrophic throughout the period of analysis (up to 12 months) showed significantly more cytokeratin 16 expression at 3 months, when compared tither with normal scars or with hypertrophic scars that became normal after 12 months, Staining for Ki-67 antigen, a marker for cell proliferation, revealed an increase in basal keratinocyte proliferation rate in 3-month-old hypertrophic scars compared with non-hypertrophic scars. After 12 months, this difference had disappeared completely and the number of cycling basal cells had returned to normal values, Three-month-old hypertrophic scars showed more acanthosis than non-hypertrophic scars of the same age, irrespective of whether they remained hypertrophic or became normal scars. After 12 months, this difference was no longer present, Staining for various heparan sulphate proteoglycan epitopes revealed that restoration of the basal membrane was incomplete at 3 months, but was complete at 12 months with respect to this component, No differences in the expression of several components of the basal membrane zone (heparan sulphate proteoglycan, laminin, tenascin) were noted between hypertrophic and non-hypertrophic scars, These data show that in the early phase of hypertrophic scarring, epidermal abnormalities are found compared with normal wound healing, In addition, early (3 months) epidermal abnormalities are associated with the clinical outcome at 12 months, These findings raise the possibility that the epidermal compartment is involved in the pathogenic process, (C) 1998 John Wiley & Sons, Ltd.

Although in the last few years in general the biocompatibility of biomaterials has significantly improved, unwanted tissue reactions are often observed resulting in early resorption of the biomaterial, loosening of the implant or in a chronic (immunologic) response. From immunologic studies it is known that inflammatory reactions can be modulated by use of (anti) growth factors or anti-inflammatory drugs. Before this can be employed the role of individual factors (humoral and cellular) involved in the inflammatory reaction against biomaterials has to be studied. In this part of the study the role of macrophages is studied with and without depletion by use of the liposomes-mediated macrophage suicide technique. Crosslinked dermal sheep collagens were used as biodegradable test materials. The results showed that macrophage depletion increases vascularization, and decreases the infiltration of granulocytess into the collagens. The foreign body reaction, i.e. the infiltration of macrophages and giant cells was significantly inhibited, resulting in a strongly delayed degradation time of the biomaterials. However, macrophage depletion did not inhibit attraction of fibroblasts and even resulted in increased formation of autologous rat-collagen, which improved the biocompatibility and the function of the biomaterials as a tempory scaffold.

Although in the last few years in general the biocompatibility of biomaterials has significantly improved, unwanted tissue reactions are often observed resulting in early resorption of the biomaterial, loosening of the implant or in a chronic (immunologic) response. From immunologic studies it is known that inflammatory reactions can be modulated by use of (anti) growth factors or anti-inflammatory drugs. Before this can be employed the role of individual factors (humoral and cellular) involved in the inflammatory reaction against biomaterials has to be studied. In this part of the study the role of macrophages is studied with and without depletion by use of the liposomes-mediated macrophage suicide technique. Crosslinked dermal sheep collagens were used as biodegradable test materials. The results showed that macrophage depletion increases vascularization, and decreases the infiltration of granulocytess into the collagens. The foreign body reaction, i.e. the infiltration of macrophages and giant cells was significantly inhibited, resulting in a strongly delayed degradation time of the biomaterials. However, macrophage depletion did not inhibit attraction of fibroblasts and even resulted in increased formation of autologous rat-collagen, which improved the biocompatibility and the function of the biomaterials as a tempory scaffold.

In order to establish the number, the viability and the developmental potential of GABAergic neurons present in dopaminergic ventral mesencephalic (VM) grafts from embryonic rat, we have studied the survival and development of these neurons in culture. The GABAergic fraction demonstrated a highly disproportionate survival in culture in relation to other VM neurons resulting in a drastic change in the neuronal composition of the dissociated VM grafts. The occurrence of a similar gradual dominance of GABAergic neurons at the site of intracerebral implantation, may affect the development of grafted dopaminergic VM neurons and their interaction with host striatal cells.

Bu çalışma, 2000 yılında İzmir'de düzenlenen 22. Ulusal Türk Plastik Rekonstrüktif ve Estetik Cerrahi Derneği Kongresi'nde bildiri olarak sunulmuştur. Since several experimental and clinical studies demonstrated the chondrogenic potential of perichondrium there has been great interest in examining factors that might promote neochondrogenesis from perichondrium. Human amniotic fluid contains hyaluronic acid. growth factors and extracellular macromolecules, and may. therefore, have a stimulating effect on cartilage regeneration. This experimental Study investigated the effect of human amniotic fluid on cartilage regeneration from rabbit ear perichondrial flaps, using 96 ears of 48 New Zealand young rabbits. A perichondrial flap was elevated and a cartilage defect measuring 20 min x 15 nim was created on the dorsum of each ear, then the perichondrial flap was Sutured in place. The ears were divided into two groups according to the solution injected underneath the perichondrial flap. The right ears, which were injected with 0.2 ml human amniotic fluid, formed the experimental group, and the left ears, which were injected with 0.2 ml saline, formed the control group. Macroscopic and histological progression of neochondrogenesis were evaluated at 2, 4 6 and 8 weeks after surgery. Macroscopically, the cartilage in the experimental group was generated quickly and had a similar appearance to the surrounding cartilage tissue, whereas in the control group minimal cartilage formation was observed at 4 weeks. Histologically, the neocartilage was significantly thicker in the experimental group than in the control group at 8 weeks (P < 0.05, Student's t-test). It can be concluded that human amniotic fluid enhances new cartilage formation from rabbit car perichondrial flaps. The preventive effect of human amniotic fluid on scar formation and the rich content of growth factors and extracellular matrix precursors may play a role in this result. (C) 2002 The British Association of Plastic Surgeons.

In both larval and adult urodele amphibians, limb blastema formation requires the presence of an adequate nerve supply. In previous research, we demonstrated that the hindlimb of early Xenopus laevis larvae formed a regeneration blastema even when denervated, while the denervated limb of late larvae did not. We hypothesized that the nerve-independence was due to the autonomous synthesis of a mitogenic neurotrophic-like factor by undifferentiated limb bud cells. In this paper, we demonstrate that fgf-2 mRNA is present in larval limb tissues and that its level is correlated to the extent of mesenchymal cells populating the limb: in early limbs, fgf-2 mRNA is present at high levels all over the limb, while, in late limbs, the fgf-2 expression is low and detectable only in the distal autopodium. After denervation, fgf-2 mRNA synthesis increases in amputated early limbs but not in amputated late limbs. The implantation of anti-FGF-2 beads into amputated early limbs hardly lowers the mitotic activity of blastema cells. However, FGF-2 beads implanted into the blastema of late limbs prevent the denervation-induced inhibition of mitosis and oppose blastema regression. Our data indicate that FGF-2 is a good candidate for the endogenous mitogenic factor responsible for blastema formation and growth in amputated and denervated early limbs. However, in amputated late limbs, the very limited fgf-2 expression is not sufficient to promote blastema formation in the absence of nerves. (C) 2001 Academic Press. ispartof: Developmental biology vol:231 issue:2 pages:436-446 ispartof: location:United States status: published

In this paper we address the linking of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (FGF-2) to intracellular signaling molecules in oligodendrocyte progenitors. It is demonstrated that both growth factors activate downstream targets similar to those shown for protein kinase C (PKC) activation. Yet, neither the arrest of terminal oligodendrocyte differentiation nor the proliferation induced by PDGF or FGF-2 can be antagonized by inhibition of PKC. Rather, p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, and pp70 S6 kinase were found to be necessary for the mitogenic activity of PDGF and FGF-2. Paradoxically, these kinases were also necessary for the onset of oligodendrocyte differentiation in control cells. In addition, cAMP-dependent kinase A (PKA) activation inhibited the mitogenic response of oligodendrocyte progenitors to FGF-2. Taken together, the molecular mechanism that controls oligodendrocyte lineage progression is operated by at least two signal pathways, which interfere either with proliferation and/or differentiation of oligodendrocyte progenitors.

The factors that regulate the ontogeny and differentiation of C1 adrenergic neurons located in the rostral ventrolateral medulla (RVLM) are completely unknown. In the present study, we have investigated the effects of a number of neurotrophic factors on the survival of E18-19 rat C1 adrenergic neurons in culture. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to study the expression of tyrosine hydroxylase (TH), an enzyme present in all catecholaminergic neurons, and of phenylethanolamine N-methyltransferase (PNMT), the final enzyme in the synthesis of adrenalin, as markers for the C1 RVLM neurons. Our results show that GDNF, CNTF BDNF, NT-3 and NT-4/5 increase the number of TH-immunoreactive neurons surviving in vitro. The effects of NGF, TGF beta and bFGF were not significant. The E18-19 CI neurons appeared to loose their ability to express PNMT in culture as examined with immunocytochemistry and RT-PCR, and none of the tested neurotrophic factors was able to sustain or induce this expression. Our results indicate that the adrenergic phenotype of C1 neurons, or the survival of these neurons, is determined by environmental factors other than the neurotrophic factors examined in this study. (C) 1999 Elsevier Science B.V. All rights reserved.

Fibrin or a fibrinous exudate can facilitate angiogenesis in many pathological conditions. In vitro, the outgrowth of capillary-like structures in fibrin can be mimicked by exposing human microvascular endothelial cells (hMVECs) to an angiogenic growth factor and tumor necrosis factor (TNF)-alpha. Urokinase-type plasminogen activator (u-PA) and plasmin activities are required for this angiogenic process. This study focuses on the role and localization of the u-PA receptor (u-PAR) in newly formed microvascular structures. The u-PAR-blocking monoclonal antibody (MAb) H-2 completely inhibited the formation of capillary-like tubular structures induced by exposure of hMVECs to basic fibroblast growth factor and TNF-alpha. This was accompanied by a several-fold increase in u-PA accumulation in the conditioned medium. The effect of MAb H-2 was not caused by blocking cellular activation by u-PA/u-PAR interaction, as the amino-terminal fragment (ATF) of u-PA, which also activates u-PAR, prevented tube formation. In addition, the inhibition by MAb H-2 was not due to an effect of the antibody on u-PAR-vitronectin binding. These data show that inhibition of tube formation can be caused not only by inhibition of u-PA or plasmin activities but also by unavailability of the u-PAR for cell-bound proteolysis. Immunohistochemical analysis showed that in in vitro angiogenesis u-PAR and u-PA were localized on the invading, tube-forming hMVECs and not on the endothelial cells that are located on top of the fibrin matrix. u-PAR and u-PA were also prominently expressed on endothelial cells of neovessels present in an atherosclerotic plaque. These data may give more insight into the role of u-PAR in repair-associated angiogenesis.

Cathepsin B and L (cysteine proteases), urokinase- and tissue-type plasminogen activators (serine proteases), and type-1 inhibitor are involved in gastric mucosal injury. We determined tissue protease levels in duodenal ulcer and their relationship to ulcer phase, bleeding tendency, Helicobacter pylori infection, and use of H2-blockers.Endoscopic biopsies of antral and duodenal mucosa were obtained from 61 patients with active or healed duodenal ulcer and control subjects. Antigen concentrations were measured by ELISA.Significantly higher levels of cathepsins, urokinase-type plasminogen activator, type-1 inhibitor, and significantly lower levels of tissue-type plasminogen activator were found in active ulcers. Bleeders had the highest cathepsins and urokinase-type plasminogen activator levels. Higher levels of urokinase-type plasminogen activator, cathepsin B, and type-1 inhibitor were observed in Helicobacter pylori-positive patients.Our results suggest that impaired fibrinolysis (tissue-type plasminogen activator), intramucosal proteases (cathepsins), tissue remodeling, and angiogenesis (urokinase-type plasminogen activator and type-1 inhibitor) are involved in duodenal ulcer formation and healing.

In addition to its pivotal role in the coagulation cascade, thrombin is mitogenic for fibroblasts and endothelial cells, and activates a number of inflammatory cells including monocytes and T-lymphocytes. To determine if other immune functions are modulated by thrombin and if this modulation is direct or indirect, we investigated whether highly purified human α-thrombin affects natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. Thrombin enhanced NK cell-mediated cytotoxicity by more than 60% and enhanced IL-2 production and NK 3.3 cell responsiveness to IL-2. Unexpectedly, thrombin and the receptor activating “tethered ligand” domain of the thrombin receptor (TRP-7:SFLLRNP) inhibited LAK cell-mediated cytotoxicity by 50%. DIP–thrombin (a proteolytically inactive form of α-thrombin) had no inhibitory activity, suggesting that proteolytic activation of thrombin receptor is requisite for inhibition. These results indicate that cell-mediated cytotoxicity may be enhanced by thrombin through a mechanism involving stimulation of cytokine production and NK cell responsiveness, but that activation of thrombin receptor may also inhibit cytotoxic effects of LAK cells. The role of this dual regulation in processes of cell surveillance, wound healing, and inflammation remains to be determined.

Although in the last few years in general the biocompatibility of biomaterials has significantly improved, unwanted tissue reactions are often observed resulting in early resorption of the biomaterial, loosening of the implant or in a chronic (immunologic) response. From immunologic studies it is known that inflammatory reactions can be modulated by use of (anti) growth factors or anti-inflammatory drugs. Before this can be employed the role of individual factors (humoral and cellular) involved in the inflammatory reaction against biomaterials has to be studied. In this part of the study the role of macrophages is studied with and without depletion by use of the liposomes-mediated macrophage suicide technique. Crosslinked dermal sheep collagens were used as biodegradable test materials. The results showed that macrophage depletion increases vascularization, and decreases the infiltration of granulocytess into the collagens. The foreign body reaction, i.e. the infiltration of macrophages and giant cells was significantly inhibited, resulting in a strongly delayed degradation time of the biomaterials. However, macrophage depletion did not inhibit attraction of fibroblasts and even resulted in increased formation of autologous rat-collagen, which improved the biocompatibility and the function of the biomaterials as a tempory scaffold.

In order to establish the number, the viability and the developmental potential of GABAergic neurons present in dopaminergic ventral mesencephalic (VM) grafts from embryonic rat, we have studied the survival and development of these neurons in culture. The GABAergic fraction demonstrated a highly disproportionate survival in culture in relation to other VM neurons resulting in a drastic change in the neuronal composition of the dissociated VM grafts. The occurrence of a similar gradual dominance of GABAergic neurons at the site of intracerebral implantation, may affect the development of grafted dopaminergic VM neurons and their interaction with host striatal cells.