Multi-organ phenotypes in mice lacking latent TGFβ binding protein 2 (LTBP2)

Abstract

Background: Latent TGFβ binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFβ, although it may interfere with the function of other LTBPs or interact with other signaling pathways.

Results: Here, we investigate mice lacking Ltbp2 (Ltbp2^-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype.

Conclusions: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.

Keywords: LTBP2; LTBPs; TGFβ; fibroblasts; myofibroblasts.

Figure 1:. Ltbp2 expression in mouse tissues

(A) Relative expression of Ltbp2 in selected tissues (Lattin et al. ref. 60). (B-D) Single cell expression analysis from selected tissues from multiple previously published data sets (UMAP). (B) Lung cell expression (Tsukui et al. ref. 64) showing highest expression in myofibroblasts (MyoFB). (C) Skin cell expression (Phan et al ref. 69) showing highest expression in fibroblasts(FB). (D) Aorta expression (Lin et al. ref. 65) showing highest expression in the vascular smooth muscle (VSM) and myofibroblasts. (E-F) Expression of Ltbp2 in skeletal tissue. (E) Periosteal expression (Julien et al. ref. 62) showing high expression in fibroblasts and immature osteoblasts (OB). (F) Endocortical expression (Ayturk et al. ref. 63) showing highest expression in Cxcl12-abundant-reticular (CAR) cells. CB, ciliary bodies;; SM, smooth muscle; CC, cerebral cortex; BM, bone marrow; Peri, pericytes; Krt, keratinocytes; Mac, macrophage; AlvFB, alveolar fibroblast; Neut, neutrophil; B, B-cell; NK, natural killer; ASM, airway smooth muscle; LipoFB, lipofibroblast; AdvFB, adventitial fibroblast; FB. Fibroblast; Mono, monocyte; Endo, endothelial; OC, osteoclast.

Figure 2:. Generation of the Ltbp2 KO mouse line

(A) Diagram showing the wild type Ltbp2 allele, the targeted allele, and the allele generated after removing the neo cassette. (B) PCR genotyping. (C) PCR for the Neo gene. (D) Ltbp2 expression(qPCR) in Mouse Embryonic Fibroblasts (MEFs) showing absence in expression in MEFs derived from Ltbp2^−/− mice. Open bar, Ltbp2^+/+; Shaded bar, Ltbp2^−/−, p<0.001.

Figure 3:. Body fat and size comparison of control and Ltbp2^−/− mice

(A, B) Growth curve of male (n= 8 Ltbp2^+/+ control and 8 Ltbp2^−/−) and female (n= 5 Ltbp2^+/+ control and 8 Ltbp2^−/−) mice. Significant differences in weight are observed beginning at ∼30 weeks of age in males. (C, D) Body fat % of Ltbp2^+/+ control and Ltbp2^−/− mice at 12, 16, and 44 weeks as determined from DEXA scans. (E) Size comparison of male mice at 40 weeks of age also showing loss of back hair in Ltbp2^−/− mice. (F) Epididymal fat pad size comparison at 24 weeks of age. (G) Skeletal area of male (blue) and female (pink) WT and Ltbp2^−/− mice at 12, 16, and 44 weeks of age as determined by DEXA scans. Open bar, Ltbp2^+/+ ; Shaded bar, Ltbp2^−/−. ns, not significant; * p<0.05; ** p<0.01.

Figure 4:. Analysis of tail length and pliability of control and Ltbp2^−/− mice

(A) X-ray (DEXA) images of Ltbp2^+/+ and Ltbp^−/− mice at 12 weeks of age. (B) Number of caudal vertebrae in 12-week-old of Ltbp2^+/+ and Ltbp^−/− mice. (C) Tail length of Ltbp2^+/+ and Ltbp^−/− mice measured in X-ray images. (D) Increased tail flexibility in Ltbp2^−/− compared to WT mice. (E) Tail tip bleeding time. Open bar, Ltbp2^+/+; Shaded bar, Ltbp2^−/−. *** p<0.001.

Figure 5:. Cortical bone analysis of control and Ltbp2^−/− mice

(A) Immunostaining showing high LTBP2 expression in the periosteum (tibia), CB indicated cortical bone. (B) Immunostaining showing LTBP2 expression around the trabecular region, proximal (tibia), TB indicates trabecular bone (C-H) Analysis of mid-femur cortical bone by micro-CT. (C) Representative micro-CT images of mid-femur sections from 4-week-old Ltbp2^+/+ control and Ltbp2^−/− mice. (D-H) Quantitation of micro-CT measurements of cortical bone from 4, 16, and 24 week old Ltbp2^+/+ control and Ltbp2^−/− mice: (D) Cortical thickness (Ct.Th), (E) Cortical area (Ct.Ar), (F) Medullary area (Ma.Ar), (G) Principal moment of inertia (pMOI), and (H) tissue mineral density (TMD). Open bar, Ltbp2^+/+ control; Shaded bar, Ltbp2^−/−. ns, not significant; ** p<0.01; *** p<0.001.

Figure 6:. Trabecular bone analysis of control and Ltbp2^−/− mice

(A) Representative micro-CT images of proximal femur trabecular sections from 4-week-old Ltbp2^+/+ control and Ltbp2^−/− mice. (B-F) Quantitation of micro-CT measurements of proximal femur sections from 4, 16, and 24 week old Ltbp2^+/+ control and Ltbp2^−/− mice: (B) Metaphyseal bone volume/total volume ratio (BV/TV), (C) Volumetric Bone Mineral Density (vBMD), (D) Trabecular Thickness (Tb.Th), (E) Trabecular Number (Tb.N), (F) Trabecular Separation (Tb.Sp). (G, H) Representative H&E (G) and TRAP (H) staining of the distal tibia. (I) Quantitation of number of osteoclasts per bone surface (N.Oc/BS) determined from Bioquant analysis of TRAP-stained histologic sections. Open bar, Ltbp2^+/+ control; Shaded bar, Ltbp2^−/−. (J) Relative expression of Rankl and Opg in 4-week Ltbp2^+/+ control and Ltbp2^−/− mice. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

Figure 7:. Bone length and biomechanics of control and Ltbp2^−/− mice

(A) A schematic of 3-point bending. (B) Femur length of 24-week-old male mice measured with digital calipers. (C-E) Analysis of 3-pt bending of femurs from 4-, 16-, and 24-week Ltbp2^+/+ control and Ltbp2^−/− male mice: (C) Bone stiffness, (D) Maximum load, (E) Work to fracture, (F) Indentation distance determined from biodent analysis of 44-week-old male mice. No significant difference was observed. Open bar, Ltbp2^+/+ control; Shaded bar, Ltbp2^−/−. ns, not significant; * p<0.05, ** p<0.01; *** p<0.001.

Figure 8:. Analysis of skin phenotypes of control and Ltbp2^−/− mice

(A) Immunohistochemical staining for LTBP2 from back skin of 24-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice. (B) Representative H&E staining of 24-week-old back skin from Ltbp2^+/+ control and Ltbp2^−/− male mice. (C) Skin thickness determined by laser scanning of 10-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice. (D) Cases of ulcerative dermatitis in Ltbp2^+/+ control and Ltbp2^−/− mice. (E) Transmission electron micrographs of back skin from 24-week-old Ltbp2^+/+ control and Ltbp2^−/ male mice. Boxed regions are enlarged in E’. (F) Histogram quantification of collagen fiber sizes from 24-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice (n= 3). (G) Hydroxyproline content of back skin of 4 and 16-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice. (H) Skin ultimate failure force determined by strip testing of 10-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice. Open bar, Ltbp2^+/+ control; Shaded bar, Ltbp2^−/−. * p<0.05; ** p<0.01.

Figure 9:. Blood pressure and cardiovascular assessment of control and Ltbp2^−/− mice

Cardiovascular function of 12-week-old control (Ltbp2^+/+), Ltbp2^−/+, and Ltbp2^−/− male mice. (A) Systolic blood pressure (SBP). (B) Diastolic blood pressure (DBP). (C) Mean arterial blood pressure (MBP). (D) Pulse pressure (PP). (E) Heart rate (HR). (F) Heart weight/Body weight ratio (HW/BW). (G) Aortic compliance. (H) Carotid artery compliance. Open bar, control (Ltbp2^+/+); Light shaded bar, Ltbp2^−/+; Dark shaded bar, Ltbp2^−/−. No significant differences were observed in any measurement with respect to genotype.

Figure 10:. Analysis of lung of control and Ltbp2^−/− mice

(A) Immunofluorescent staining for LTBP2 from lungs from 10-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice. LTBP2 expression is highest in airway epithelial cells. (A’) Enlarged detail of checkered region of 10A (B) Representative H&E-stained sections from 10-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice. (C) Mean Linear Intercept measurement from 10-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice. (D) Airway dynamic compliance of 30-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice with methacholine. (E) Airway resistance of 30-week-old Ltbp2^+/+ control and Ltbp2^−/− male mice with methacholine. Open bar, Ltbp2^+/+ control; Shaded bar, Ltbp2^−/−. No significant differences were observed in any measurement with respect to genotype.