Dermal stiffness but not collagen maturation is altered in the dermis of Loxl2-depleted and overexpressing transgenics.

<p>A: Herovici staining, enabling visualisation of immature collagen fibres (blue) and mature collagen (pink/purple). B: Collagen density quantification of Loxl2-KO, Loxl2-KI and control skin samples at the indicated postnatal ages. Collagen density values were calculated as collagen pixel per total tissue. C: Quantification of B-CHP signal. The data shown are means ± SD; n = 7 (Control), n = 5 (Loxl2-KO), n = 2 (Loxl2-KI) at P21 and n = 4 (Control), n = 6 (Loxl2-KO), n = 2 (Loxl2-KI) at P120. ns; not significant. D,E: AFM measurments of P21 old dorsal dermis. Experimental strategy with a representative 50 <b>μ</b>m² scan area showing the Young’s Modulus <i>E</i> distribution (D). Quantification of Young’s modulus <i>E</i> distribution in control, Loxl2-KO and Loxl2-KI dermis at P21. The mean is shown below (n = 4 biological replicates per genotype with 3 scan areas per sample).</p

Expression of Lox family members murine and human dermis.

<p>A,B: qRT-PCR analysis of Lox, Loxl1, Loxl2, Loxl3 and Loxl4 mRNA expression during mouse development (A) and in FACS-sorted fibroblast subpopulations (papillary fibrbroblasts, CD26⁺ SCA1^-; reticular fibroblasts, CD26^- SCA1⁺) (B) from PDGFRαH2BeGFP mice. C: Expression of LOX family members in human foetal and adult dermis. Details of tissue samples are shown in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199679#pone.0199679.s005" target="_blank">S1 Table</a>). ND = no signal detectable.</p

Fibroblast subpopulations are not affected by Loxl2 deletion or overexpression.

<p>A: Immunostaining for α-SMA (green), DLK1 (red) and CD26 (white). B: Immunostaining for LRIG1 (red) and SCA1 (white). Nuclei are labelled with DAPI (blue). Scale bars: 100 <b>μ</b>m. C: Mean immunofluorescence quantification of DLK1 (left panel) and LRIG1 (right panel) in the upper and lower dermis (n = 2 for P2 Loxl2-KI, n = 3 for all others). Data are means ±SD. Note the high expression of DLK1 in the lower, reticular dermis and LRIG1 in the upper, papillary dermis at P2, which are down regulated with age.</p

Loxl2 deletion or overexpression do not affect skin response to TPA.

<p>A,B: Quantification of dermal cells in the upper, papillary (A) and lower, reticular (B) dermis upon TPA treatment. The data shown are means ± SD; n = 2 (untreated control), n = 6 (Control TPA), n = 2 (Loxl2-KO TPA), n = 3 (Loxl2-KI TPA). C: Hematoxylin and eosin (H&E) staining. D: Herovici staining. E: Picrosirius red staining visualised in polarised light. Scale bars: 100 μm.</p

Loxl2 ablation and overexpression are not compensated by Lox family members and do not alter dermal histology, thickness or cell density.

<p>A: qPCR analysis of Lox family member mRNA expression in Loxl2-KO and Loxl2-KI mice at P2. (n = 4 for control and Loxl2-KO; n = 2 for Loxl2-KI). B: Hematoxylin and eosin (H&E) staining. Scale bars: 100 <b>μ</b>m. C,D: Dermal thickness from basal membrane to hypodermis (C) and of the hypodermis (D). E,F: Total dermal cell number in upper, papillary (E) and lower, reticular dermis (F). (n = 2 for P2 and P120 Loxl2-KI, n = 3 for all others).</p

Coronary Artery Disease–Associated Locus on Chromosome 9p21 and Early Markers of Atherosclerosis

Background— Genome-wide association studies have recently identified a locus on chromosome 9p21 that influences risk of coronary artery disease (CAD). The effect of the locus on early markers of atherosclerosis is unknown. We examined its association with carotid intima-media thickness (CIMT) and brachial flow-mediated dilatation (FMD). Methods and Results— We genotyped 2277 individuals, age 24 to 39 years, from the Cardiovascular Risk in Young Finns Study with CIMT and FMD measurements and 1295 individuals, age 46 to 76 years, from the Health 2000 Survey with CIMT for rs1333049, the chromosome 9p21 variant showing the strongest association with CAD. Both mean and maximum CIMT were significantly higher (P<0.001) in the older subjects of the Health 2000 Survey compared with the Young Finns Study. However, there was no association of the rs1333049 genotype with either mean or maximum CIMT at either age (P=0.959 and 0.977 for the 2 phenotypes in the Young Finns Study and P=0.714 and 0.725 in the Health 2000 Survey). Similarly, there was no association of the locus with variation in FMD in the Young Finns cohort (P=0.521). Conclusions— The chromosome 9p21 locus does not influence CAD risk through a mechanism that also affects CIMT or induces early changes in FMD. We examined the association with carotid intima-media thickness and brachial flow mediated dilatation of the recently identified susceptibility locus for coronary artery disease on chromosome 9p21. We found no evidence that the risk variant affects either of these early markers of atherosclerosis, suggesting an alternate mechanism for its effect on risk of CAD