Utility of hyaluronan oligomers and transforming growth factor-beta1 factors for elastic matrix regeneration by aneurysmal rat aortic smooth muscle cells

Abstract

The progression of aortic aneurysms (AAs) is typically associated with an activated smooth muscle cell (SMC) phenotype, diminished density of mature medial elastic fibers, and an elevated presence of matrix-degrading enzymes, which ultimately leads to vessel rupture. Currently, no surgical or nonsurgical methods are available to regress aneurysms via regeneration of new elastic matrices, particularly because of inherently poor elastin synthesis by adult vascular cells and absence of tools to stimulate the same. We seek to address this void in this study. We recently showed 0.2 microg/mL of hyaluronan oligomers and 1 ng/mL of transforming growth factor-beta1 (termed elastogenic factors) to dramatically enhance elastin synthesis and matrix formation by healthy aortic SMCs. In this study, the effect of these factors, alone or together, on suppressing procalcific and elastolytic activities of aneurysmal vascular cells, and improving their elastin matrix synthesis and assembly is examined. Periadventitial injury with calcium chloride was used to induce AAs in rats, and approximately 45% increase in aortic diameter was observed after 4 weeks. Aneurysmal SMCs isolated from these AA segments produced higher levels of inflammatory markers matrix metalloproteinases-2 and 9 elastase activity and calcific deposits, while synthesizing significantly less collagen, tropoelastin, and matrix elastin proteins over a 3-week culture period, relative to healthy SMCs. While hyaluronan oligomers alone significantly suppressed aneurysmal cell proliferation and promoted 20-50% increases in collagen and elastin synthesis (p < 0.01), transforming growth factor-beta1 alone had no effect on cellular proliferation and elastin synthesis. However, provision of factors together resulted in significantly higher amounts of collagen/elastin protein synthesis and crosslinking, by upregulating lysyl oxidase and desmosine. Compared to their individual contributions, the factors together were highly effective in minimizing the release of inflammatory enzymes, and encouraging elastic fiber formation. Since elastic matrix amounts were one order of magnitude lower than that observed with healthy cells, even upon elastogenic stimulation at doses optimized previously for healthy cells, increased doses are likely required and must be reoptimized for diseased cells. Despite this, the results point to the potential utility of these elastogenic factors in regenerating elastic matrices within AAs.

FIG. 1.

Representative images of abdominal rat aortae before calcium chloride (CaCl₂) treatment (A) and 28 days postinjury (B), when significant elastin degradation was observed. The aortae showed a local diameter increase of 45%. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Histological analysis of rat aortae shown 38 days posttreatment with 0.5 M CaCl₂ and 0.5 M NaCl (treatment control). The solutions were applied for 15 min, postadventitially. Hematoxylin and eosin–stained sections of l2-treated aortae showed extensive breakdown of elastic lamellar structures (pink; see arrows) in the tunica media layer, in contrast to those within NaCl-treated aortae, which remained intact. These results were confirmed by comparison of elastin (black structures) within Verhoeff-Van Gieson–stained sections, and using immunofluorescence (note holes in green-fluorescing elastic lamellae). Von Kossa staining revealed presence of calcific deposits within aortae treated with CaCl₂, but not NaCl, confirming the induction of an abnormal, disease-like microenvironment in the former case. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Significant rounding of the smooth muscle cells (SMCs) isolated from the aneurysmal segment was observed on day 1 (A) and more spindle-shaped by day 7 (B). SMCs isolated from healthy aortae showed more spread morphology on day 7 of seeding (C). The cells were stained with SMC-α actin to confirm the SMC phenotype (D). Color images available online at www.liebertonline.com/ten.

FIG. 4.

(A) Proliferation ratios of aneurysmal SMC cultures supplemented with oligomers alone, transforming growth factor-β (TGF-β) alone, or cues together. Data shown represent mean ± SD of DNA content of cell layers after 21 days of culture, normalized to initial seeding density, and further normalized to aneurysmal control cultures that received no additives (n = 3/case). (B) Effects of oligomers alone, TGF-β alone, and cues together, on total collagen synthesis by aneurysmal SMCs. Data shown (mean ± SD) are normalized to cellular DNA content at 21 days of culture and represented as fold change in protein production relative to aneurysmal controls (n = 3/case). p < 0.05 represents significant differences from controls (*).

FIG. 5.

Effects of oligomers alone, TGF-β alone, and both cues together, on tropoelastin (A), alkali-soluble (B), crosslinked matrix elastin (C), and total elastin (D) produced by aneurysmal SMCs. Data shown (mean ± SD) are normalized to cellular DNA content at 21 days of culture and represented as fold change in protein production relative to aneurysmal controls (n = 3/case). p < 0.05 represents significant differences from controls (*).

FIG. 6.

(A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis/Western blot analysis of tropoelastin and Lysyl oxidase (LOX) proteins within the pooled medium of aneurysmal cultures at the end of 21 days. Data shown represent mean ± SD of three repeats/case and are shown normalized to controls. (B) Desmosine amounts measured in test cell layers were normalized to corresponding DNA amounts (ng/ng), and further a similar ratio obtained for the healthy nonadditive controls. Comparable trends were observed for the desmosine/DNA density and respective insoluble matrix elastin/DNA for selected cases. *Represents significance of differences in values relative to cultures of healthy additive-free cells, deemed for a p value <0.05.

FIG. 7.

Gel zymography analysis revealed the presence of matrix metalloproteinases-2 (A) and matrix metalloproteinases-9 (B) within aneurysmal cultures treated with or without cues. Data were shown normalized to the respective values observed in healthy nonadditive treatment controls (n = 3/case). (C) Elastase enzyme activity within aneurysmal SMC cultures treated with oligomers alone, TGF-β alone or together with hyaluronan (HA) oligomers. Data are shown normalized to the corresponding values in healthy nonadditive cultures (n = 3/case). Von Kossa staining images of untreated aneurysmal cell layers (D) or those treated with TGF-β alone (E), oligomers alone (F), or cues together (G), and healthy cell layers (H). Significant calcific deposits were evident in control cultures and those treated with TGF-β alone or oligomers alone, while cultures that received cues together showed a decrease in the calcific mineralization, almost to levels exhibited in healthy SMC layers. *Indicates significance of differences relative to healthy, additive-free control cell cultures, deemed for p < 0.05.

FIG. 8.

Immunodetection of elastin, fibrillin, and LOX within control and test cell layers after 21 days of culture. For each labeled protein, images of each of the treatment control (no additives) and test cell layers were collected under identical image capture settings and were identically brightness/contrast enhanced for the purpose publication. An average of 15 random locations were imaged on each of the three replicate cell layers/case/set. An increase in matrix elastin, fibrillin, and LOX is evident in aneurysmal cultures that received both TGF-β and HA oligomers, relative to nonadditive aneurysmal SMC cultures (treatment controls). Cultures that received TGF-β alone or oligomers alone showed relatively less intense fluorescence relative to cultures receiving both cues. Lack of fluorescence in cultures untreated with primary antibodies (negative immunofluorescence controls) confirms lack of nonspecific binding of the fluorophore-conjugated secondary antibody. Scale bar: 150 μm. Color images available online at www.liebertonline.com/ten.

FIG. 9.

Representative transmission electron microscopy images of 21-day-old aneurysmal SMC layers cultured additive-free aneurysmal SMC layers [treatment control; (A) 50000×; (B) 100000×] and those cultured with both TGF-β and HA oligomers [(C) 50000×; (D) 100000×]. Aggregating amorphous elastin clumps leading to the formation of elastin fibers can be clearly seen in aneurysmal cell cultures treated with the factors (C, D), while aneurysmal SMC cultures that received no additives (A, B) showed amorphous elastin deposits with sparse fiber formation. In all cultures, immunogold labeling showed presence of fibrillin microfibrils (black dots), indicative of normal mechanisms of elastic matrix assembly.