Aortic carboxypeptidase-like protein enhances adipose tissue stromal progenitor differentiation into myofibroblasts and is upregulated in fibrotic white adipose tissue

Figures

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  Fig 1. ACLP expression is repressed during adipogenesis. A, Scheme of time course for 10T1/2 adipogenic differentiation. B, 10T1/2 fibroblasts were induced to undergo adipogenesis with DMII on day 0. Protein was harvested on days -2, 0, +2, +4 and +6. Day +8 cells were fractionated into differentiated adipocytes (A) and the non-differentiated 10T1/2 fibroblasts (ND) and harvested for protein. Protein expression was analyzed using SDS-PAGE and Western blot with antibodies against ACLP, α-SMA, FABP4, adiponectin (AdipoQ) and cyclophilin A. Protein expression was quantified by densitometry normalized to CypA expression and all samples were compared to day 0 cells or mature adipocyte fraction. *, p < 0.05 versus control, one way ANOVA with post hoc Tukey’s test for day -2, 0, +2, +4 and +6 samples and one sample t-test for fractionated day +8 samples.

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  Fig 2. ACLP signaling represses adipogenesis. A, Scheme of 10T1/2 adipogenesis and rACLP treatment. B, 10T1/2 fibroblasts were treated with 30 nM rACLP on days -2, -1, 0, +2 and +4 and induced to undergo adipogenesis with DMII on day 0. Protein was harvested on day +6 and analyzed by SDS-PAGE and Western blot with antibodies against ACLP, collagen, α-SMA, AdipoQ, FABP4, PPARγ and cyclophilin-a (CypA). PPARγ analysis was performed in separate experiments. Protein expression was quantified by densitometry normalized to CypA expression and relatively compared to control cells. *, p < 0.05 versus control, one sample t-test for all values. C, 10T1/2 fibroblasts were treated with 30 nM rACLP on days -2, -1, 0, +2 and +4 and induced to undergo adipogenesis with DMII on day 0. On day +6 cells were fixed, stained, imaged and quantified with Oil Red O dye (n = 3). Data were normalized relative to untreated controls. *, p < 0.05 versus paired control, one sample t-test. Data presented are expressed as mean ± SD. The scale bar represents 2 mm.

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  Fig 3. ACLP inhibition of adipogenesis and enhancement of myofibroblast differentiation is dependent on TGFβ receptor activity. A, Scheme of 10T1/2 fibroblast adipogenesis with SB431542 and rACLP treatment. B, 10T1/2 progenitors were concurrently treated with 30 nM rACLP and vehicle control or 1 μM SB431542, a TGFβ receptor kinase inhibitor, on days -2, -1, 0, +2 and +4 as well as induced to undergo adipogenesis on day 0. Protein was harvested on day +6 and analyzed by SDS-PAGE and Western blot with antibodies against ACLP, α-SMA, AdipoQ, FABP4 and CypA. Protein expression was quantified by densitometry normalized to CypA expression and relatively compared to untreated control cells (n = 3). One way ANOVA with post hoc Tukey’s test for all values (* p < 0.05, ** p < 0.005). Data presented are expressed as mean ± SD.

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  Fig 4. ACLP expression increases in the stromal vascular fraction from diet induced fibrotic epididymal adipose tissue. A, epididymal adipose tissue from C57BL6/J male mice on low fat diet or high fat diet for 16 weeks (n = 3 for each diet group) was excised and fixed with methyl Carnoy, stained with picrosirius red and imaged using brightfield (left panels) and under polarized light (right panels). B, Tissue sections of epididymal adipose tissue following low fat diet or high fat diet were immunostained for ACLP and perilipin and counterstained with DAPI. Data are representative of parallel 10 μm sections. Closed arrowhead indicates vasculature. Open arrowhead indicates peri-cellular staining. Scale bar represents 100 μm. C, Epididymal adipose tissue from C57BL6/J male mice on low fat diet or high fat diet for 16 weeks (n = 3 for each diet group) was excised, enzymatically digested, fractionated based on buoyancy and then protein lysates were generated of each population. Whole stromal vascular fraction lysate was analyzed by SDS-PAGE and Western blot with antibodies against ACLP, α-SMA, desmin, SM22, CRP2 and CypA. Protein expression was quantified by densitometry normalized to CypA…

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  Fig 5. CD45- SVF derived from fibrotic adipose tissue is the source of ACLP expression. A, a scheme of isolating SVF CD45-, SVF CD45+ and adipocyte populations from epididymal adipose tissue. B, CD45- SVF, CD45+ immune and adipocyte populations were isolated from epididymal adipose tissue from C57BL6/J male mice on high fat diet for 16 weeks (n = 4) and mRNA levels were determined by qPCR analysis of fibrosis, inflammation and adipocyte markers. * p < 0.05, one way ANOVA with post hoc Tukey’s test for all values. Data presented are expressed as mean ± SD.

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  Fig 6. High fat diet reduces adipogenic potential of total SVF. A, Epididymal adipose tissue from C57BL6/J male mice on low fat diet or high fat diet for 16 weeks (n = 3 for each group) was excised, enzymatically digested and depleted of CD45+ cells. CD45- SVF cells were then cultured for 24 hours in 10% FBS DMEM and subsequently media was collected and analyzed by SDS-PAGE and Western blot with antibodies against ACLP. B, CD45- SVF cells were isolated as above and cultured for 10 days in 10% FBS DMEM, DMII was given on day 2 and cell lysates were harvested on day 10. Cell lysates were analyzed by SDS-PAGE and Western blot with antibodies against adiponectin, perilipin and FABP4. Protein expression was quantified by densitometry normalized to CypA expression and relatively compared to low fat diet cells (n = 3). * p < 0.05, Students t-test used for all values. C, SVF cells were isolated as above and were cultured (100,000 cells/well (12 well)) until confluent, and then induced to undergo adipogenesis with DMII 2 days after confluent. Oil Red O staining and imaging was performed 8 days following adipogenic induction (n = 3). * p < 0.05, Students t-test used for value. The scale bar…

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  Fig 7. Recombinant ACLP inhibits adipogenesis and enhances myofibroblast differentiation in human adipose stromal cells. A, Scheme of time course for adipogenesis of hASC. B, hASC derived from subcutaneous adipose depots were induced to undergo adipogenesis with CDM on day 0. Protein was harvested on days 0, +1, +2, +8 and +15. Protein expression was analyzed using SDS-PAGE and Western blot with antibodies against ACLP, α-SMA, FABP4 and cyclophilin-A. C, Omental hASC were induced to undergo adipogenesis with CDM on day 0. Protein was harvested on days 0, +1, +2, +8 and +15. Protein expression was analyzed using SDS-PAGE and Western blot with antibodies against ACLP, α-SMA, FABP4 and cyclophilin-A. D, Scheme of adipogenesis for hASC and rACLP treatment. E, Subcutaneous hASC were treated with 30 nM rACLP on days -2, -1 and 0 and induced to undergo adipogenesis with CDM on day 0 and +4. Protein was harvested on day +15 and analyzed by SDS-PAGE and Western blot with antibodies against collagen, α-SMA, perilipin, FABP4 and CypA (n = 3). F, Subcutaneous hASC were treated with 30 nM rACLP on days -2, -1, and 0 and induced to undergo adipogenesis with CDM on day 0 and +4. On day +15 cells were…