Fig 2: Expression of neurotrophin receptors in human aortic smooth muscle cells (HASMCs). A, Representative agarose gel to demonstrate typical levels of tropomyosin-related kinase (Trk) isoform A (TrkA; 401 bp product), B (TrkB; 575 bp product), and C (TrkC; 455 bp product) and of p75NTR (192 bp product) mRNA expression in HASMCs. Human umbilical vein endothelial cells (HUVECs) were examined in parallel for comparison. B, Flow cytometry analysis of HASMCs using smooth muscle actin (SMA) to visualize smooth muscle cells (SMCs) as well as 2 different antibodies against TrkB (H-181 or F-1) or against an epitope within the C-terminus present in all Trk isoforms (pan-Trk). The percentage of positive cells (in 1×106 total cells) is indicated within the graphs. C, Immunofluorescence confocal microscopy analysis of HASMCs stained with antibodies directed against TrkB (green signal). Scale bars indicate 20 µm. Phalloidin was used to visualize the cytoskeleton (red signal) and DAPI (4',6-diamidino-2-phenylindole) to stain cell nuclei (blue signal). Immunohistochemical analysis of TrkB expression in internal mammary artery and aorta using antibodies against SMA (D) or CD68 (E) as markers for SMCs or macrophages, respectively, on immediately neighboring sections. Scale bars indicate 50 µm. FITC indicates fluorescein isothiocyanat; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IMA, internal mammary artery; and MW, molecular weight.

Fig 3: Effects of brain-derived neurotrophic factor (BDNF) stimulation on human aortic smooth muscle cells (HASMCs) and importance of protein tyrosine phosphatase 1B (PTP1B).HASMCs were cultivated until subconfluency, incubated with PTP1B inhibitor (50 µmol/L) or dimethyl sulfoxide (DMSO) for 1 hour before being stimulated with recombinant human BDNF (10 and 20 ng/mL) for an additional 24 hours. RNA was isolated and changes in mRNA expression of cyclin D1 (CCND1; A), smooth muscle a-actin (ACTA2; B), smooth muscle myosin heavy chain (MYH11; C), COL1A1 (D), tropomyosin kinase B (TrkB; E), and p75NTR (F) examined using real-time polymerase chain reaction. *P<0.05, **P<0.01, and ****P<0.0001 vs DMSO; # P<0.05 and ## P<0.01 vs PTP1B alone and § P<0.05, §§ P<0.01, §§§ P<0.001, and §§§§ P<0.0001 vs BDNF (10 ng/mL), as determined using 1-way ANOVA (A, D, E, and F) or Kruskall-Wallis test (B and C) followed by multiple comparisons tests. G, Confocal microscopy images after immunostaining of HASMCs with antibodies against p75NTR (green signal). Scale bars indicate 20 µm. DAPI indicates 4',6-diamidino-2-phenylindole.

Fig 4: Hypothetical signaling of perivascular neurotrophins in vascular smooth muscle cells (SMCs) during atherosclerosis and the effects of protein tyrosine phosphatase 1B (PTP1B) or its inhibition.Chronically increased expression of brain-derived neurotrophic factor (BDNF) as well as other adipokines (eg, leptin), inflammatory mediators (eg, tumor necrosis factgor a [TNF-a]), and counterregulatory phosphatases (eg, PTP1B), as observed in perivascular adipose tissue surrounding the aortic root and coronary arteries of patients with advanced atherosclerosis, is associated with downregulation of the BDNF receptor on SMCs. Negative regulation of tropomyosin kinase (Trk) B signaling by PTP1B may contribute to atherosclerosis by altering the effects of BDNF on SMC proliferation, migration, differentiation, or collagen production. Inhibition of PTP1B upregulates the low-affinity neurotrophin receptor p75NTR and inhibits the effects of BDNF on SMCs.

Fig 5: Expression of neurotrophin receptors in human arterial vessels.mRNA levels of tropomyosin-related kinase (Trk) isoform A (TrkA; A), B (TrkB; B), and C (TrkC; C) and of the low affinity receptor p75NTR (D) were determined using reverse transcription polymerase chain reaction in tissue samples of the A. mammaria interna (IMA; n=9) and the aortic wall (n=7). The results of the statistical analysis are indicated within the graphs. A representative agarose gel showing results in paired samples of IMA and aorta from 2 patients are shown (E). Paraffin-embedded cross-sections through the IMA or aorta were stained using Masson trichrome (MTC) stain (upper row; smooth muscle cells in red) or antibodies against TrkB (middle row; positive immunosignals in brown) (F). The results after omission of the first antibody (negative control; bottom row) are also shown. Scale bars indicate 50 µm. GAPDH indicates glyceraldehyde 3-phosphate dehydrogenase.