Journal: Aging cell

Article Title: Tendinosis develops from age- and oxygen tension-dependent modulation of Rac1 activity.

doi: 10.1111/acel.12934

Figure Lengend Snippet: FIGURE 1 Aged and diseased tendon display altered cellular morphology and extracellular matrix production in vivo. (a–c) Intra‐operative photographs of (a) 84‐year‐old female patient undergoing fibrocartilage pulley release for recalcitrant flexor tendinosis (aged), (b) 42‐year‐old female patient undergoing tendinosis excision prior to reconstruction (tendinosis), (c) 30‐year‐old female patient undergoing primary tendon repair after acute laceration (young). Forcep tip delineates the flexor tendon. (d–f) Alcian blue (AB) histopathologic staining of (a‐c), respectively. Bar = 20 μm. (g–i) Alizarin red (AR) histopathologic staining of (a‐c), respectively. Bar = 20 μm. (j–u) Immunohistochemical staining of aged (j,m,p,s), tendinosis (k,n,q,t), and young (l,o,r,u) patient samples as in (a–c) for: col1α2 (j‐l), col2α1 (m–o), colX (p–r) and HIF‐1α (s–u). Bar = 20 μm. (v) Quantitation of immunoblot of collagen 1α2 and collagen 2α1 expression from young asymptomatic tendon (Young) or aged diseased tendon (Aged). *p < 0.05, n = 4. (w, x) FTIR of young asymptomatic tendon (Young) or aged diseased tendon (Aged tendinotic). (w) Protein:collagen ratio. (x) Mineral:collagen ratio. Spectrophotometric absorbance as shown below quantitation with absorbance scale to the right. *p < 0.05, n = 4

Article Snippet: Primary immunostaining was performed using antibodies against collagen 1α2 (gift from Dr. A. Fertala), collagen 2α1 (OriGene), collagen X (Abcam), and HIF‐1α (Abcam).

Techniques: In Vivo, Staining, Immunohistochemical staining, Quantitation Assay, Western Blot, Expressing

Journal: Aging cell

Article Title: Tendinosis develops from age- and oxygen tension-dependent modulation of Rac1 activity.

doi: 10.1111/acel.12934

Figure Lengend Snippet: FIGURE 2 Aged tenocytes display altered cellular morphology and extracellular matrix production in vitro. Human tenocytes from aged (Aged), young tendinotic (Tendinotic), and young asymptomatic (Young) patients were cultured at low (1k/cm2 aged a‐b; 5k/cm2 young tendinotic c, d and young e, f) cell densities in hypoxic (1% O2; a, c, e) or normoxic conditions (21% O2; b, d, f) over twelve weeks. Shown are phase contrast micrographs of representative cells from week 2 (wk2), week 8 (wk8), and week 12 (wk12). Bar = 50 μm. (g–j) Brightfield photomicrographs of alizarin red stained aged (g, h) or young asymptomatic (i, j) tenocytes in hypoxic (1% O2, g, i) or normoxic (21% O2, h, j) conditions at week 12. (k) Spectrophotometric quantitation of calcification (mg/dL) as detected by alizarin red staining of aged (Aged) or young asymptomatic (Young) tenocytes cultured in hypoxic (Hypoxia, 1% O2) or normoxic (Normoxia, 21% O2) conditions at low (Low, 1k/cm2 for aged, 5k/cm2 for young asymptomatic) or high (High, 25k/cm2 for aged, 50k/cm2 for young asymptomatic) cell densities and harvested at week 12 in culture. *p < 0.05, n = 4 (l–q) qRT–PCR expression of aged (red), young tendinotic (green), or young asymptomatic (blue) tenocyte (C1, Scl, Tm, C3), fibrochondrocyte (C2), and mineralized fibrochondrocyte (CX) markers over a 12‐week timecourse, hypoxic (1% O2) as compared to normoxic (21% O2) conditions, at cell densities: 1k = 1 × 103cells/cm2; 5k = 5 × 103 cells/cm2; 25k = 25 × 103 cells/cm2; 50k = 50 × 103cells/cm2 (not available for aged tenocytes). C1 = col1α2; C2 = col2α1; C3 = col3; CX = colX; Scl = scleraxis; Tm = tenomodulin

Article Snippet: Primary immunostaining was performed using antibodies against collagen 1α2 (gift from Dr. A. Fertala), collagen 2α1 (OriGene), collagen X (Abcam), and HIF‐1α (Abcam).

Techniques: In Vitro, Cell Culture, Staining, Quantitation Assay, Quantitative RT-PCR, Expressing

Journal: Aging cell

Article Title: Tendinosis develops from age- and oxygen tension-dependent modulation of Rac1 activity.

doi: 10.1111/acel.12934

Figure Lengend Snippet: FIGURE 3 Hypoxia regulates the aged tenocyte phenotype via modulating Rac1 Activity. Human tenocytes from aged (a, d) young tendinotic (b, e) and young asymptomatic (c, f) patients were cultured at low density (aged at 1 × 103 cells/cm2; young tendinotic and young asymptomatic at 5 × 103 cells/cm2) in hypoxia (1% O2; (H)) or normoxia (21% O2; (n)) over twelve weeks and assayed for Rac1 (a, b, c) and RhoA (c, d, e) activity. Immunoblots were quantitated using ImageJ software. Error bars = SEM. n = 3. Representative Western blots as shown

Article Snippet: Primary immunostaining was performed using antibodies against collagen 1α2 (gift from Dr. A. Fertala), collagen 2α1 (OriGene), collagen X (Abcam), and HIF‐1α (Abcam).

Techniques: Activity Assay, Cell Culture, Western Blot, Software

Journal: Aging cell

Article Title: Tendinosis develops from age- and oxygen tension-dependent modulation of Rac1 activity.

doi: 10.1111/acel.12934

Figure Lengend Snippet: FIGURE 5 Differential, coordinated Rac1 and RhoA activity direct the human tenocyte phenotype. (a) Immunoblot of RhoA activity in young tenocytes at low density (5 × 103 cells/cm2) cultured in normoxic (n; 21% O2) or hypoxic (H; 1% O2) conditions, without or with Rac1 inhibitor NSC23766 (NSC) harvested after one week. Error bars = SEM, n = 4. *p < 0.05. Representative blot shown. (b–e) Young tenocytes cultured in normoxic conditions (21% O2) at low (5 × 103cells/cm2), medium (25 × 103 cells/cm2), and high (50 × 103 cells/cm2) cell densities in the absence of drug (no drug), presence of ROCK inhibitor Y‐27632 (Y27), Rac1 inhibitor NSC23766 (NSC), dominant negative Rac1 (RacN), or RhoA (RhoN), constitutively active Rac1 (RacL), or RhoA (RhoL), without or with ROCK inhibitor Y‐27632 (/Y27) or Rac1 inhibitor NSC23766 (/NSC). Tenocytes were harvested after one week and analyzed for col1α2 (b), col3 (c), col2α1 (d), or colX (e) expression via qRT– PCR. Error bars = SEM, n = 4. ***p < 0.001. (f–q) Brightfield photomicrographs of young asymptomatic (“Young,” f–i) young tendinotic (“Tend,” j–m) or aged (“Aged,” n‐q) human tenocytes cultured in hypoxic (1% O2; (H)) or normoxic (21% O2) conditions in the presence (NSC) or absence of Rac1 inhibitor NSC23766, and harvested after one week for acid βgalactosidase senescence assay. Bar = 50 μm. (r) Quantitation of βgalactosidase‐positive staining cells as in (f–q) at time of harvest. Error bars = SEM, n = 4. *p < 0.05

Article Snippet: Primary immunostaining was performed using antibodies against collagen 1α2 (gift from Dr. A. Fertala), collagen 2α1 (OriGene), collagen X (Abcam), and HIF‐1α (Abcam).

Techniques: Activity Assay, Western Blot, Cell Culture, Dominant Negative Mutation, Expressing, Quantitative RT-PCR, Quantitation Assay, Staining

Journal: Aging cell

Article Title: Tendinosis develops from age- and oxygen tension-dependent modulation of Rac1 activity.

doi: 10.1111/acel.12934

Figure Lengend Snippet: FIGURE 6 Rac1 inhibition in human tendon explants recreates the aged diseased tendon phenotype. Young diseased tendon (“Tendinotic,” a, b, j, k) or asymptomatic (“Young,” c–h, l–o) tendon explants cultured in normoxic conditions (21% O2; a, c, j, l), normoxic conditions with Rac1 inhibitor NSC23766 (NSC, b, d, k, m), hypoxia (1% O2; e, n), or hypoxia with NSC23766 (f, g, h, o) harvested after three months, stained for H&E (a–h) or via immunohistochemistry (j–o) for col1α2 (C1), col2α1 (C2), colX (CX), or HIF‐1α (HIF1α). Magnification of explants at 4x after routine H&E staining, with close‐up at 10x. Bar = 20 μm. n = 4. Representative explants shown. (i, j) FTIR of protein:collagen ratio (i) or mineral:collagen ratio (j) of explants as above. Error bars = SEM, n = 4. *p < 0.05

Article Snippet: Primary immunostaining was performed using antibodies against collagen 1α2 (gift from Dr. A. Fertala), collagen 2α1 (OriGene), collagen X (Abcam), and HIF‐1α (Abcam).

Techniques: Inhibition, Cell Culture, Staining, Immunohistochemistry