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Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction.

Scioli MG, Lo Giudice P, Bielli A, Tarallo V, De Rosa A, De Falco S, Orlandi A - PLoS ONE (2015)

Bottom Line: A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression.Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction.The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine and Prevention, Anatomic Pathology, University of Tor Vergata, Rome, Italy.

ABSTRACT

Background: Impaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery.

Methods and results: We investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM) expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction.

Conclusion: PLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological targeting of endothelial dysfunction may represent a promising tool for the treatment of delayed wound healing or chronic ulcers.

No MeSH data available.


Related in: MedlinePlus

PLC ameliorates blood flow recovery.(A) Representative diagram of rat skin flap. On the left, the proximal (P), medial (M), and distal (D) segments. Center, representative laser doppler images of blood flow before (basal) and just after flap elevation (T0). Low or no blood perfusion is displayed in dark blue, and the highest perfusion level is displayed in red. Right, it was reported the means ± SEM of blood perfusion relative units (RU) in proximal, medial and distal portions of the flap before (baseline) and just after flap creation (T0). (B, C) Representative laser Doppler images of blood flow (B) and time course (C) of in skin flap in vehicle- (closed symbols) and PLC-treated (open symbols) rats. PLC was administered daily, dissolved in tap water at the dose of 100mg/kg. Points are mean ± SEM of 15 rats, in relative units of blood perfusion at 2, 4, 6 and 8 days after flap elevation. (C) Left, blood perfusion in distal portion of flap (repeated measures ANOVA: * indicates p< 0.05, PLC-treated vs vehicle-treated rats). Center, blood perfusion in medial portion of flap (ANOVA:** indicates p< 0.001,PLC-treated vs vehicle-treated rats). Right, blood perfusion in proximal portion of flap (ANOVA:* and ** indicate p< 0.05 and p< 0.001, respectively, PLC-treated vs vehicle-treated rats). (D,E) Representative images of flap skin and necrotic area measurement after 8 days. Necrotic area (black) is clearly demarcated from survival regions. (F) Values represent mean ± SEM of 15 rats. Repeated measures ANOVA: treatment, p< 0.0001; Dunnett’s test: * and ** indicate p< 0.001 and p< 0.0001, respectively. (F) Bar graph showing ROS levels, inversely proportional to fluorescence intensity (F.I), in rat skin flap. Values represent mean ± SEM of 15 rats. t-Student: * indicates p< 0.05. (G) iNOS protein expression and NO level (expressed in optical density, OD), in rat skin flap. Values represent mean ± SEM of 15 rats. t-Student: * indicates p< 0.05. Abbreviation: α-tub, α-tubulin.
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pone.0140697.g001: PLC ameliorates blood flow recovery.(A) Representative diagram of rat skin flap. On the left, the proximal (P), medial (M), and distal (D) segments. Center, representative laser doppler images of blood flow before (basal) and just after flap elevation (T0). Low or no blood perfusion is displayed in dark blue, and the highest perfusion level is displayed in red. Right, it was reported the means ± SEM of blood perfusion relative units (RU) in proximal, medial and distal portions of the flap before (baseline) and just after flap creation (T0). (B, C) Representative laser Doppler images of blood flow (B) and time course (C) of in skin flap in vehicle- (closed symbols) and PLC-treated (open symbols) rats. PLC was administered daily, dissolved in tap water at the dose of 100mg/kg. Points are mean ± SEM of 15 rats, in relative units of blood perfusion at 2, 4, 6 and 8 days after flap elevation. (C) Left, blood perfusion in distal portion of flap (repeated measures ANOVA: * indicates p< 0.05, PLC-treated vs vehicle-treated rats). Center, blood perfusion in medial portion of flap (ANOVA:** indicates p< 0.001,PLC-treated vs vehicle-treated rats). Right, blood perfusion in proximal portion of flap (ANOVA:* and ** indicate p< 0.05 and p< 0.001, respectively, PLC-treated vs vehicle-treated rats). (D,E) Representative images of flap skin and necrotic area measurement after 8 days. Necrotic area (black) is clearly demarcated from survival regions. (F) Values represent mean ± SEM of 15 rats. Repeated measures ANOVA: treatment, p< 0.0001; Dunnett’s test: * and ** indicate p< 0.001 and p< 0.0001, respectively. (F) Bar graph showing ROS levels, inversely proportional to fluorescence intensity (F.I), in rat skin flap. Values represent mean ± SEM of 15 rats. t-Student: * indicates p< 0.05. (G) iNOS protein expression and NO level (expressed in optical density, OD), in rat skin flap. Values represent mean ± SEM of 15 rats. t-Student: * indicates p< 0.05. Abbreviation: α-tub, α-tubulin.

Mentions: Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg; Siegfried Ltd, Zofingen, Switzerland). The design and surgical technique for construction of the rat dorsal skin flap were previously described [28,30,31]. Briefly, dorsal surface was shaved and a 10 x 3 cm caudally based dorsal skin flap was elevated from the scapulas to the iliac crest (Fig 1A), then sutured back into its original site with stainless steel wound clips. During measurements, body temperature was maintained constant at 37°C by using a thermoregulator (BM 70002, Biomedica Mangoni, Pisa, Italy) and blood pressure checked by using a piezoelectric tail-cuff pulse transducer, as reported [32]. Rats were identified by numbers; the blood pressure technician was not aware of the experimental grouping. Special care was taken to monitor their complete recovery from anesthesia, and vital parameters. No analgesia was administered during the post-surgery recovery period. Rats were randomized into two groups (n = 15); the first group received PLC (Sigma-Tau SpA, Pomezia, Italy) at the dose of 100 mg/kg/day in the drinking water, the second group only water (vehicle). The PLC concentration in the water was adjusted on the bases of body weight and water intake, daily measured and checked for one week before the beginning of the treatment and during the experiment. Revascularization was quantified by measuring the extent of survival flap by serial assessment of blood flow [33] using a scanning laser Doppler perfusion imaging (PIM II System Laser Doppler Perfusion Imager [LDPI]; Lisca/Perimed, Järfälla-Stockholm, Sweden) before (baseline), immediately after surgery (day 0),and after 2, 4, 6, and 8 days under light anesthesia by s.c pentobarbital injection (40 mg/kg). Scanned color-coded image representing blood flow distribution displayed on the monitor were divided into three equal portions: proximal (near the iliac crest), medial at the centre of the flap, and distal at the scapula (Fig 1A). Analysis of mean perfusion was performed using the manufacturer's operational software (LDPIwin) and normalized on baseline values. After the last blood flow recording, flaps were removed by cutting the margin and animals sacrificed by a lethal dose of pentobarbital. Flap and necrotic areas were traced onto the same transparent plastic sheet and planimetrically measured in blind (SigmaScan Pro5 Images Measurement Software), as reported[34]. The necrotic area in the distal portion of the skin flap was easily demarcated by gross observation [30].


Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction.

Scioli MG, Lo Giudice P, Bielli A, Tarallo V, De Rosa A, De Falco S, Orlandi A - PLoS ONE (2015)

PLC ameliorates blood flow recovery.(A) Representative diagram of rat skin flap. On the left, the proximal (P), medial (M), and distal (D) segments. Center, representative laser doppler images of blood flow before (basal) and just after flap elevation (T0). Low or no blood perfusion is displayed in dark blue, and the highest perfusion level is displayed in red. Right, it was reported the means ± SEM of blood perfusion relative units (RU) in proximal, medial and distal portions of the flap before (baseline) and just after flap creation (T0). (B, C) Representative laser Doppler images of blood flow (B) and time course (C) of in skin flap in vehicle- (closed symbols) and PLC-treated (open symbols) rats. PLC was administered daily, dissolved in tap water at the dose of 100mg/kg. Points are mean ± SEM of 15 rats, in relative units of blood perfusion at 2, 4, 6 and 8 days after flap elevation. (C) Left, blood perfusion in distal portion of flap (repeated measures ANOVA: * indicates p< 0.05, PLC-treated vs vehicle-treated rats). Center, blood perfusion in medial portion of flap (ANOVA:** indicates p< 0.001,PLC-treated vs vehicle-treated rats). Right, blood perfusion in proximal portion of flap (ANOVA:* and ** indicate p< 0.05 and p< 0.001, respectively, PLC-treated vs vehicle-treated rats). (D,E) Representative images of flap skin and necrotic area measurement after 8 days. Necrotic area (black) is clearly demarcated from survival regions. (F) Values represent mean ± SEM of 15 rats. Repeated measures ANOVA: treatment, p< 0.0001; Dunnett’s test: * and ** indicate p< 0.001 and p< 0.0001, respectively. (F) Bar graph showing ROS levels, inversely proportional to fluorescence intensity (F.I), in rat skin flap. Values represent mean ± SEM of 15 rats. t-Student: * indicates p< 0.05. (G) iNOS protein expression and NO level (expressed in optical density, OD), in rat skin flap. Values represent mean ± SEM of 15 rats. t-Student: * indicates p< 0.05. Abbreviation: α-tub, α-tubulin.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608702&req=5

pone.0140697.g001: PLC ameliorates blood flow recovery.(A) Representative diagram of rat skin flap. On the left, the proximal (P), medial (M), and distal (D) segments. Center, representative laser doppler images of blood flow before (basal) and just after flap elevation (T0). Low or no blood perfusion is displayed in dark blue, and the highest perfusion level is displayed in red. Right, it was reported the means ± SEM of blood perfusion relative units (RU) in proximal, medial and distal portions of the flap before (baseline) and just after flap creation (T0). (B, C) Representative laser Doppler images of blood flow (B) and time course (C) of in skin flap in vehicle- (closed symbols) and PLC-treated (open symbols) rats. PLC was administered daily, dissolved in tap water at the dose of 100mg/kg. Points are mean ± SEM of 15 rats, in relative units of blood perfusion at 2, 4, 6 and 8 days after flap elevation. (C) Left, blood perfusion in distal portion of flap (repeated measures ANOVA: * indicates p< 0.05, PLC-treated vs vehicle-treated rats). Center, blood perfusion in medial portion of flap (ANOVA:** indicates p< 0.001,PLC-treated vs vehicle-treated rats). Right, blood perfusion in proximal portion of flap (ANOVA:* and ** indicate p< 0.05 and p< 0.001, respectively, PLC-treated vs vehicle-treated rats). (D,E) Representative images of flap skin and necrotic area measurement after 8 days. Necrotic area (black) is clearly demarcated from survival regions. (F) Values represent mean ± SEM of 15 rats. Repeated measures ANOVA: treatment, p< 0.0001; Dunnett’s test: * and ** indicate p< 0.001 and p< 0.0001, respectively. (F) Bar graph showing ROS levels, inversely proportional to fluorescence intensity (F.I), in rat skin flap. Values represent mean ± SEM of 15 rats. t-Student: * indicates p< 0.05. (G) iNOS protein expression and NO level (expressed in optical density, OD), in rat skin flap. Values represent mean ± SEM of 15 rats. t-Student: * indicates p< 0.05. Abbreviation: α-tub, α-tubulin.
Mentions: Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg; Siegfried Ltd, Zofingen, Switzerland). The design and surgical technique for construction of the rat dorsal skin flap were previously described [28,30,31]. Briefly, dorsal surface was shaved and a 10 x 3 cm caudally based dorsal skin flap was elevated from the scapulas to the iliac crest (Fig 1A), then sutured back into its original site with stainless steel wound clips. During measurements, body temperature was maintained constant at 37°C by using a thermoregulator (BM 70002, Biomedica Mangoni, Pisa, Italy) and blood pressure checked by using a piezoelectric tail-cuff pulse transducer, as reported [32]. Rats were identified by numbers; the blood pressure technician was not aware of the experimental grouping. Special care was taken to monitor their complete recovery from anesthesia, and vital parameters. No analgesia was administered during the post-surgery recovery period. Rats were randomized into two groups (n = 15); the first group received PLC (Sigma-Tau SpA, Pomezia, Italy) at the dose of 100 mg/kg/day in the drinking water, the second group only water (vehicle). The PLC concentration in the water was adjusted on the bases of body weight and water intake, daily measured and checked for one week before the beginning of the treatment and during the experiment. Revascularization was quantified by measuring the extent of survival flap by serial assessment of blood flow [33] using a scanning laser Doppler perfusion imaging (PIM II System Laser Doppler Perfusion Imager [LDPI]; Lisca/Perimed, Järfälla-Stockholm, Sweden) before (baseline), immediately after surgery (day 0),and after 2, 4, 6, and 8 days under light anesthesia by s.c pentobarbital injection (40 mg/kg). Scanned color-coded image representing blood flow distribution displayed on the monitor were divided into three equal portions: proximal (near the iliac crest), medial at the centre of the flap, and distal at the scapula (Fig 1A). Analysis of mean perfusion was performed using the manufacturer's operational software (LDPIwin) and normalized on baseline values. After the last blood flow recording, flaps were removed by cutting the margin and animals sacrificed by a lethal dose of pentobarbital. Flap and necrotic areas were traced onto the same transparent plastic sheet and planimetrically measured in blind (SigmaScan Pro5 Images Measurement Software), as reported[34]. The necrotic area in the distal portion of the skin flap was easily demarcated by gross observation [30].

Bottom Line: A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression.Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction.The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedicine and Prevention, Anatomic Pathology, University of Tor Vergata, Rome, Italy.

ABSTRACT

Background: Impaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery.

Methods and results: We investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM) expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction.

Conclusion: PLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological targeting of endothelial dysfunction may represent a promising tool for the treatment of delayed wound healing or chronic ulcers.

No MeSH data available.


Related in: MedlinePlus