Proinflammatory signals and the loss of lymphatic vessel hyaluronan receptor-1 (LYVE-1) in the early pathogenesis of laminin alpha2-deficient skeletal muscle

Abstract

Congenital muscular dystrophy type 1A, a severe neuromuscular disease characterized by early-onset muscle weakness and degeneration, is caused by insufficient levels of laminin α2 (LAMA2) in the basal lamina surrounding muscle fibers and other cells. A better understanding of the molecular mechanisms leading to muscle loss is needed to develop therapeutic interventions for this disease. Here, the authors show that inflammation is an early feature of pathogenesis in Lama2-deficient mouse muscle, indicated by elevated expression of tenascin C in the endomysium around muscle fibers, infiltration of macrophages, and induction of the inflammatory cytokines tumor necrosis factor α (TNFα) and IL-1β. In addition, the expression of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), a specific marker for lymphatic vessel endothelial cells, is dramatically reduced early in Lama2-deficient muscle pathogenesis. LYVE-1 expression, which is inhibited by TNFα, is also decreased in muscles undergoing degeneration due to dystrophin deficiency and cardiotoxin damage. LYVE-1 expression thus provides a useful biomarker to monitor the onset of muscle pathogenesis, likely serving as an indicator of inflammatory signals present in muscles. Together, the data show that inflammatory pathways are activated in the earliest stages of Lama2-deficient disease progression and could play a role in early muscle degeneration.

Figure 1.

Pathological features are evident in Lama2-deficient mice within 7 days after birth. (A) Lama2^-/- mice are of normal size at birth but are smaller than are Lama2-expressing siblings by 7 days of age. Body mass at 1 and 7 days of age is shown for wild-type Lama2^+/+ (solid bars), Lama2^+/- (shaded bars), and Lama2^-/- (white bars) mice. The two age groups represent two separate cohorts of mice, n≥12 mice for each data point (see text). Data shown as mean ± SE, *p<0.002. (B) Tenascin-C (TN-C), a marker of inflammation, is dramatically upregulated in Lama2^-/- quadriceps muscle at 7 days of age. Muscles immunostained for TN-C expression show deposition of TN-C in the endomysium surrounding muscle fibers in patches or foci within the muscle, including regions where loss of muscle integrity is not apparent morphologically. There is no TN-C in endomysium of wild-type muscle fibers at this age (scale bars=100 µm).

Figure 2.

Lymphatic vessel endothelial hyaluronan receptor–1 (LYVE-1) is specifically expressed in lymphatic vessels and is dramatically reduced in vessels of Lama2-deficient muscle. (A) Lymphatic vessels and blood vessels within skeletal muscle tissue can be distinguished by the expression of LYVE-1 and CD31 (PECAM-1). Shown are quadriceps muscles from a 3-day-old Lama2-expressing mouse double immunostained for LYVE-1 and CD31. The hyaluronan receptor LYVE-1 (green) is specifically expressed in lymphatic vessel endothelial cells. CD31, a pan-endothelial marker, is expressed by vascular endothelial cells found in blood capillary vessels (red) but is only expressed at low levels in lymphatic vessel endothelial cells and is often not detectable except in larger trunk lymphatic vessels where expression of both markers can be evident (arrows; scale bar=100 µm). (B) LYVE-1 expression in lymphatic vessels is dramatically reduced in 1- and 2-week-old Lama2(-) muscles compared with those of normal Lama2(+) mice. Quadriceps muscles from Lama2(-) (Lama2^-/-) and Lama2(+) (Lama^{+/+ or +/-}) mice were immunostained using antibodies for LYVE-1 (green) or CD31 (red). The pattern of CD31 expression in blood vessels is not affected by Lama2 deficiency (scale bars=100 µm).

Figure 3.

The loss of lymphatic vessel endothelial hyaluronan receptor–1 (LYVE-1) expression in lymphatic endothelial cells is more widespread than is tenascin C (TN-C) expression in Lama2-deficient muscles. Shown are 7-day-old hindlimb muscles from Lama2^-/- and Lama^+/+ mice double immunostained for TN-C (red) and LYVE-1 (green) expression. The top row of images is at a higher magnification than that of the lower two rows. The high- and low-magnification images are from two different Lama2^-/- mice. TN-C is normally expressed in epimysium around large groups of muscles and at sites of tension or stress (e.g., tendons, myotendinous, osteotendinous junctions). In Lama2^-/- muscles, TN-C is upregulated in the endomysium around foci of muscle fibers (small arrows). The absence of LYVE-1 in lymphatic vessels of Lama2^-/- muscles is more widespread at this age, often observed in muscles where there is no TN-C expression (arrowheads). Some Lama2^-/- muscles at this age continue to have normal patterns of LYVE-1 and TN-C expression (open arrows; scale bars=100 µm). F, fibula; T, tibia.

Figure 4.

Macrophage infiltration occurs in regions of 7-day-old Lama2^-/- muscles where tenascin C (TN-C) is expressed, whereas elevated embryonic myosin heavy chain (EmbMyHC) expression is not restricted to TN-C expressing areas. Shown are the tibialis anterior regions of 7-day-old hindlimbs from a Lama^+/+ mouse (+, top row) and three individual Lama2^-/- mice (-, three lower rows) immunostained for the proteins indicated. Neighboring tissue sections were immunostained for expression of TN-C (red), double immunostained for the macrophage marker CD11b (green) and lymphatic vessel endothelial hyaluronan receptor–1 (LYVE-1, red), or for EmbMyHC (green). Variability in the expression of these pathology markers is evident among the mutant animals, but in each case, Lama2^-/- muscle tissue exhibited localized expression of TN-C that correlated with the presence of CD11b macrophages, and there was an absence of LYVE-1 expression throughout the muscles. EmbMyHC, which is normally downregulated after birth, was low but detectable in some regions of normal muscle but remained more highly expressed in mutant muscles, and this was not restricted to muscle areas expressing TN-C (arrowheads; scale bars=100 µm). T, tibia.

Figure 5.

The expression of tenascin C (TN-C), tumor necrosis factor α and IL-1β mRNAs are upregulated in Lama2^-/- tibialis anterior muscles at 7 days of age. Quantitative PCR methods were used to measure the relative levels of mRNA in 7-day-old muscle of the indicated genotypes. The relative expression levels of each gene were normalized to glyceraldehyde-3-phosphate dehydrogenase expression and relative expression calculated in relationship to one of the wild-type samples. In each case, there was a significant (p<0.04, n=3 Lama2(-), n=4 Lama2(+)) increase in transcript levels in Lama2-deficient muscles compared with Lama2-expressing siblings. Data shown as mean ± SE.

Figure 6.

Lymphatic vessel endothelial hyaluronan receptor–1 (LYVE-1) expression is reduced in lymphatic vessels of dystrophin-deficient mdx muscles and normal muscles regenerating after cardiotoxin injury. Quadriceps muscles from 3-week-old (A, B) or 4-week-old (C, D) mice were immunostained for LYVE-1 expression. At 3 weeks, LYVE-1 expression in lymphatic vessels of affected mdx muscle (B) appeared similar to that of unaffected sibling muscle (A). At 4 weeks, mdx muscle undergoing degeneration and regeneration (D) had reduced LYVE-1 expression compared with unaffected sibling muscle (C). Regenerating tibialis anterior muscle of normal C57BL/6 mice 5 days after cardiotoxin injury exhibited reduced LYVE-1 (E) and elevated tenascin C levels (F) in regions of tissue damage and regeneration (double immunostained sample). *MyoD-hBcl-2 tg(+). (Scale bars=100 µm.)