Sortilin expression in rat aortic smooth muscle cell populations.

<p>Representative blot analysis of (A) sortilin protein expression and transcript levels (B) in normal rat aortic medial tissue and 15 days after ballooning. Sortilin and p75^NTR (C) transcripts accumulate in intimal cells obtained 15 days after injury (IT cells). The latter and normal media SMCs (mSMCs) were harvested after 3 and 6 days in sparse and confluent cultures, respectively. Representative blots and densitometric analysis (D and E) after normalization to CMR1 expression; data are mean ± SEM of three experiments. Sortilin and p75^NTR immunofluorescence (F) documents a higher intracellular sortilin and p75^NTR level in IT cells compared to mSMCs; right panel: merged images showing the prevalent co-localization of sortilin with p75^NTR; *<i>p</i><0.05. Scale bar = 25 µm.</p

Sortilin expression and apoptosis in normal and post-injury rat aortas.

<p>Anti-sortilin and anti-p75^NTR immunostainings (A) do not reveal detectable positivity in normal tunica media. Intimal thickening appears markedly sortilin, p75^NTR and proNGF positive fifteen days after ballooning, but not after forty-five days; α-SMA immunodetection goes in the opposite direction. TUNEL⁺ cells are evident in the neointima 15 days after ballooning (arrow heads)<b>.</b> Bar graphs (B) showing sortilin⁺, p75^NTR+ and TUNEL⁺ apoptotic intimal cell percentages; *<i>p</i><0.05. Scale bar = 50 µm.</p

ProNGF is a potent apoptotic inducer of IT cells.

<p>IT cells (A) express TrkB and TrkC but not TrkA transcripts, while all Trk receptors are present in mSMCs. ProNGF (B and C) is a potent apoptotic inducer of IT cells and induces a dose-dependent increase of cultured rat aortic IT cell apoptosis as measured by TUNEL in serum-free medium after 24 and 48 hours; flow cytometry (D) of rat aortic intimal cells in sub-G1 (DNA content<2N) calculated as percentages of total events (10,000 cells). Agarose gel under UV light (E) after staining with ethidium bromide showing the ladder production after blunt end linker ligation confirms the dose-dependent and higher proNGF apoptotic DNA fragmentation compared to control IT cells. Representative immunofluorescence (F) of IT cells after 24 h of proNGF (10 ng/mL) treatment shows intracellular distribution of sortilin somehow more evident in the cell membrane compartment, whereas p75^NTR localization is almost unchanged<b>.</b> Blot analysis (G and H) shows that proNGF (10 ng/mL) induces the increase of sortilin protein content only after 48 h. EMSA analysis (I) in IT cells after 24 h and 48 h of treatment with proNGF shows a significant reduction of NF-κB activity (<i>upper arrow</i>, p65/50 heterodimer; <i>lower arrow</i>, p50/50 homodimer). Data are reported as mean ± SEM of three independent experiments. *<i>p</i><0.05. Scale bar = 25 µm.</p

Sortilin immunostaining of grossly normal human young and old and atherosclerotic aorta and carotid vessels.

<p>Normal young vessels (A) do not display appreciable sortilin immunodetection; the latter is observed in old vessel intimal thickening and fatty streak and, more markedly, in fibroatheromatous plaque. Representative images (B) of serial sections of human fibroatheromatous plaque stained with Haematoxylin-Eosin (H-E), α-smooth muscle actin (α-SMA), CD68, sortilin, p75^NTR and proNGF. Diaminobenzidine as chromogen, Haematoxylin counterstaining. Scale bar = 50 µm.</p

Silencing of sortilin partially prevents proNGF-induced apoptosis of IT cells.

<p>Representative immunofluorescence (A) and densitometric analysis of blots (B) showing reduced sortilin expression in sortilin-silenced IT cells. Sortilin silencing (C) reduces 24 h proNGF-induced apoptosis. Graph (D) shows that SN50-induced NF-κB inhibition increases apoptosis of IT cells but not of mSMCs, that doesn’t further increase after successive 24 h of proNGF treatment. Data as mean ± SEM of three independent experiments. *<i>p</i><0.05. Scale bar = 25 µm.</p

PLC ameliorates cell function in serum-deprived HMVECs.

<p>(A) Real-time PCR for PlGF mRNA in serum-deprived PBS-treated (vehicle) or PLC-treated HMVECs at different times. (B) PlGF protein concentration assessed by means of ELISA in treated cells. (C, D) Real-time PCR for Flt-1 and KDR transcripts. (E, F) Real-time PCR for eNOS and iNOS mRNA. (G) NO measurement in vehicle or PLC-treated cells at different times. (H, I) Real-time PCR for VCAM-1 and ICAM-1transcripts. (J) Leukocyte adhesion assay on vehicle and PLC-treated HMVECs and (K) representative microphotographs at 12h, magnification 100X. t-Student: * and ** indicate p< 0.05 and p< 0.01, respectively. Values are expressed as mean ± SEM of three separate experiments. Abbreviations: OD, optical density; HPF, high power field.</p

PLC ameliorates mitochondrial β-oxidation in serum-deprived HMVECs.

<p>(A) FAD level (β-oxidation impairment) measured as optical density (OD) assay in basal condition (5% FBS) and serum-deprived HMVECs treated with PBS (vehicle, 12h), L-aminocarnitine (L-amino, 1μM, 12h) and/or PLC (1mM, 12h). (B) ROS level detection by dichlorodihydrofluorescein fluorescence intensity (DCF, F.I) assay in treated cells. (C) Real-time PCR for Nox4 transcripts in treated cells. (D) Leukocyte adhesion evaluation in treated cells. Data are shown as mean ± SEM. Student’s t-test: *, ** and *** indicate p< 0.05; p< 0.01 and p< 0.001. Abbreviations: OD, optical density; HPF, high power field.</p

PLC reduces oxidative stress in serum-deprived HMVECs.

<p>(A) Real-time PCR for Nox4 transcripts in serum-deprived PBS-treated (vehicle) or PLC-treated HMVECs at different times. (B) ROS level detection by dichlorodihydrofluorescein fluorescence intensity (DCF F.I). (C) FAD level (β-oxidation impairment) measured as optical density (OD) assay. t-Student: * and ** indicate p< 0.05 and p< 0.01, respectively. Values are expressed as mean ± SEM of three separate experiments. Abbreviations: OD, optical density.</p

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

<div><p>Background</p><p>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.</p><p>Methods and Results</p><p>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.</p><p>Conclusion</p><p>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.</p></div

Histomorphometric analysis of rat skin wounds granulation tissue.

<p>(A) Representative CD31, VEGF, PlGF, iNOS, Nox4 immunostaining of skin granulation tissue sections in vehicle and PLC-treated rats on day 7 wounds. Magnification 100X. (B) Columns represent the percentage of immunoreactive microvessels and stain intensity calculated with a grading system in arbitrary units. Abbreviation: a.u., arbitrary units; t-Student: *, ** and *** indicate p< 0.05, p< 0.01 and p< 0.001, respectively. Values are expressed as mean ± SEM of 15 rats.</p