Journal: International Journal of Biological Sciences

Article Title: N-Homocysteinylation of HMGB1/2 Promotes Corpus Cavernosum Endothelial Senescence in Erectile Dysfunction

doi: 10.7150/ijbs.119514

Figure Lengend Snippet: HHcy induces ED in middle-aged rats via accelerated endothelial senescence. A. Representative laser speckle contrast imaging of sodium nitroprusside (NO donor)-induced rat penile blood flow across four experimental groups (n=3): Young (control), Young+HHcy, Mid-old (natural aging), and Mid-old+HHcy. Color gradients (blue-to-red) indicate relative perfusion levels in anatomical subregions: corpus cavernosum (CC, erectile tissue), penile body (PB), and glans penis (GP). B. Representative curves of electrostimulation-induced intracavernosal pressure dynamics and hemodynamic quantification (n=5 per each group). AP: arterial pressure; ICP: intracavernosal pressure; MAP: mean arterial pressure; Total ICP: area under the ICP curve. C. Representative images and semi-quantification of Masson's trichrome-stained rat corpus cavernosum sections (n=4). Scale bars: 50 μm. D. Representative images and semi-quantification of SA-β-gal activity in rat corpus cavernosum sections (n=4). Scale bars: 50 μm. E-F. Representative immunofluorescence co-staining images and semi-quantification in different groups: E. Laminb1 (green) and Lectin (endothelium, red). F. p21 (green) and Lectin. G. UMAP projection plot of the combined single-cell transcriptomes across all experimental groups. FB (Fibroblast), EC (Endothelial cell), SMC (Smooth muscle cell). H. Stacked bar plots of single-cell composition across experimental groups. I. Pseudotemporal trajectory analysis of senescence-associated genes Cdkn1a , H2ax , and Il6 in EC subpopulations. Solid lines represent HHcy-accelerated aging, dashed lines denote natural aging. J. Violin plots of senescence-associated gene expression in EC subpopulations across groups. K. EC state transition dynamics revealed by Monocle2 pseudotime analysis. Left: Pseudotemporal trajectory of ECs showing bifurcation at Branch Point 1 (senescence initiation). Red arrows indicate HHcy-accelerated transitions from pre-aging (State 1) to senescence state (State 3). Right: Pie charts quantify the proportion of ECs in three pseudotime-defined states.

Article Snippet: Primary rat corpus cavernosum endothelial cells (CCECs) were isolated from 6-week-old male Sprague-Dawley rats using enzymatic digestion combined with mechanical extrusion, as previously optimized .

Techniques: Imaging, Control, Staining, Activity Assay, Immunofluorescence, Gene Expression

Journal: International Journal of Biological Sciences

Article Title: N-Homocysteinylation of HMGB1/2 Promotes Corpus Cavernosum Endothelial Senescence in Erectile Dysfunction

doi: 10.7150/ijbs.119514

Figure Lengend Snippet: Hcy accelerates endothelial senescence via MARS1-HTL-protein K-Hcy pathway. A. Schematic representation of Hcy-induced protein lysine-homocysteinylation (K-Hcy). B-C. Age-dependent accumulation of Hcy (B) and HTL (C) in corpus cavernosum of naturally aged rats (n=4/group). D-E. Representative immunofluorescence images (D) and semi-quantification (E) of K-Hcy in naturally aged rat penile sections. Scale bars: 100 μm. F. Representative immunofluorescence images of K-Hcy and MARS1 co-localization in human corpus cavernosum from physiological Hcy donors. Scale bars: 50 μm. G. Representative immunoblot of K-Hcy in passage 2 (P2) rat primary corpus cavernosum endothelial cells (CCECs) treated with Hcy (200 μM) or HTL (100 μM) for 12 h. H. Representative immunoblot of MARS1 and K-Hcy in CCECs across population doublings (P1, P3, P6, P9). I-J. Western blot analysis of MARS1 and senescence markers (LAMINB1, p53, p21, p16) in HCMECs with MARS1 knockdown (P10-15) (I) or MARS1 overexpression (P3-P8) (J) treated with 200 μM Hcy for 96h. Band intensities were quantified relative to the untreated group (n=3). K. Representative images and semi-quantification of SA-β-gal activity in MARS1-knockdown or -overexpression HCMECs treated with 200 μM Hcy for 96h (n=3). Scale bars: 50 μm.

Article Snippet: Primary rat corpus cavernosum endothelial cells (CCECs) were isolated from 6-week-old male Sprague-Dawley rats using enzymatic digestion combined with mechanical extrusion, as previously optimized .

Techniques: Immunofluorescence, Western Blot, Knockdown, Over Expression, Activity Assay

Journal: International Journal of Biological Sciences

Article Title: N-Homocysteinylation of HMGB1/2 Promotes Corpus Cavernosum Endothelial Senescence in Erectile Dysfunction

doi: 10.7150/ijbs.119514

Figure Lengend Snippet: N-acetylcysteine attenuates endothelial senescence and rescues erectile function in middle-aged HHcy rat by blocking K-Hcy. A. N-Acetylcysteine (NAC) decreased intracellular HTL levels induced by homocysteine. CCECs were treated with 200 μM Hcy, 100 μM HTL and 1 mM NAC for 12 h before harvesting (n=4). B. Representative images and semi-quantification of SA-β-gal activity in CCECs treated with Hcy (200 μM) and NAC (2 mM) for 96h (n=4). Scale bars: 50 μm. C. Representative laser speckle contrast imaging of sodium nitroprusside (NO donor)-induced rat penile blood flow across five experimental groups (n=3): Mid-old, Mid-old+HHcy, Mid-old+HHcy+Tadalafil (TAD), Mid-old+HHcy+NAC and Mid-old+HHcy+Tadalafil & NAC (T&N). D. Hcy and HTL levels in rat corpus cavernosum tissues across all experimental groups ( n = 4). E, I. Representative images and semi-quantification of Masson's trichrome-stained rat corpus cavernosum sections (n=4). Scale bars: 50 μm. F, J. Representative immunofluorescence images and semi-quantification of K-Hcy in rat penile sections. Scale bars: 100 μm. G, K. Representative immunofluorescence co-staining images and semi-quantification in different groups (n=4). Laminb1 (green) and Lectin (red). H, L. Representative immunofluorescence co-staining images and semi-quantification in different groups (n=4). p21 (green) and Lectin (red). Scale bars: 20 μm.

Article Snippet: Primary rat corpus cavernosum endothelial cells (CCECs) were isolated from 6-week-old male Sprague-Dawley rats using enzymatic digestion combined with mechanical extrusion, as previously optimized .

Techniques: Blocking Assay, Activity Assay, Imaging, Staining, Immunofluorescence