Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Dec 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2015 Oct 22;35(12):2647–2656. doi: 10.1161/ATVBAHA.115.306573

Postnatal Deletion of the Type II TGF-Beta Receptor in Smooth Muscle Cells Causes Severe Aortopathy in Mice

Jie Hong Hu 1,*, Hao Wei 1,*, Mia Jaffe 1, Nathan Airhart 1, Liang Du 1, Stoyan N Angelov 1, James Yan 1, Julie K Allen 1, Inkyung Kang 1, Thomas Wight 1, Kate Fox 1, Alexandra Smith 1, Rachel Enstrom 1, David A Dichek 1
PMCID: PMC4743752  NIHMSID: NIHMS730872  PMID: 26494233

Abstract

Objective

Prenatal deletion of the type II TGF-β receptor (TBRII) prevents normal vascular morphogenesis and smooth muscle cell (SMC) differentiation, causing embryonic death. The role of TBRII in adult SMC is less well studied. Clarification of this role has important clinical implications because TBRII deletion should ablate TGF-β signaling and blockade of TGF-β signaling is envisioned as a treatment for human aortopathies. We hypothesized that postnatal loss of SMC TBRII would cause aortopathy.

Approach and Results

We generated mice with either of two tamoxifen-inducible SMC-specific Cre (SMC-CreERT2) alleles and homozygous floxed Tgfbr2 alleles. Mice were injected with tamoxifen, and their aortas examined 4 and 14 weeks later. Both SMC-CreERT2 alleles efficiently and specifically rearranged a floxed reporter gene and efficiently rearranged a floxed Tgfbr2 allele, resulting in loss of aortic medial TBRII protein. Loss of SMC TBRII caused severe aortopathy including hemorrhage, ulceration, dissection, dilation, accumulation of macrophage markers, elastolysis, abnormal proteoglycan accumulation, and aberrant SMC gene expression. All areas of the aorta were affected, with the most severe pathology in the ascending aorta. Cre-mediated loss of SMC TBRII in vitro ablated both canonical and noncanonical TGF-β signaling and reproduced some of the gene expression abnormalities detected in vivo.

Conclusions

SMC TBRII plays a critical role in maintaining postnatal aortic homeostasis. Loss of SMC TBRII disrupts TGF-β signaling, acutely alters SMC gene expression, and rapidly results in severe and durable aortopathy. These results suggest that pharmacologic blockade of TGF-β signaling in humans could cause aortic disease rather than prevent it.

Keywords: Aortic Aneurysm, Aortic Dissection, Transforming Growth Factor-Beta

Transforming growth factor beta (TGF-β) signaling in cultured smooth muscle cells (SMC) or SMC precursors regulates cell growth and migration. TGF-β signaling also promotes synthesis of SMC differentiation markers and extracellular matrix proteins.1–6 Loss of SMC TGF-β signaling during development prevents SMC differentiation and matrix synthesis, causing severe vascular abnormalities and embryonic lethality.7–10 Without question, SMC TGF-β signaling is essential for normal embryogenesis. However, the role of TGF-β signaling in adult aortic SMC is less well established, and the critical question of whether SMC TGF-β signaling is salutary or pathogenic remains controversial. Salutary roles for adult SMC TGF-β signaling are suggested by animal studies showing that cardiovascular overexpression of TGF-β increases matrix synthesis and prevents aneurysm growth and that systemic blockade of TGF-β exacerbates angiotensin II-induced aortic aneurysms.11–15 In contrast, pathogenic effects of adult SMC TGF-β signaling are suggested by apparent increases in aortic SMC TGF-β signaling in association with human aortopathies (Marfan, Loeys-Dietz, Shprintzen-Goldberg syndromes, familial thoracic aortic aneurysms and dissections associated with mutations in TGF-β2) and in mouse models of these diseases.16–20 These reports, along with evidence that TGF-β antagonism achieved by injection of a neutralizing anti-TGF-β antibody could prevent aortopathy in Fbn1-deficient mice,17 spawned the hypothesis that TGF-β signaling in aortic SMC is pathogenic, and prompted clinical trials aimed at preventing aortopathy with a drug (Losartan) that is hypothesized to block TGF-β signaling. 21–23

We used genetically modified mice to test whether disruption of physiological TGF-β signaling by SMC-targeted deletion of the type II TGF-β receptor (TBRII) affects aortic homeostasis. We found that SMC TBRII loss has profound effects on aortic structure, SMC gene expression, aortic extracellular matrix accumulation, and cell proliferation. While this work was being carried out, another group reported results of similar experiments.24 Here we extend those findings, using a different conditional Tgfbr2 allele25 and two inducible SMC-specific Cre-recombinase strains (Acta2-CreERT2 and Myh11CreERT2).26, 27 We also report in vitro data suggesting that cell-autonomous alterations in SMC TGF-β signaling are the primary cause of aortopathy in mice with SMC-specific loss of TBRII. Our results reveal a critical role for adult aortic SMC TGF-β signaling and suggest a cautious approach toward pharmacologic strategies that block SMC TGF-β signaling in humans.

Material and Methods

Material and Methods are available in the online-only Data Supplement.

Results

Similar Cre Recombinase Activity in Two Lines of SMC-CreERT2 Mice

We compared the inducibility, efficiency, and tissue-specificity of Cre recombinase activity in two lines of mice with SMC-targeted inducible Cre alleles (Acta2-CreERT2 and Myh11-CreERT2). We generated mice that were hemizygous for one of these two alleles and either hemi- or homozygous for the R26R reporter allele. Mice from each line were injected with tamoxifen (Tm; n = 6) or vehicle (n = 2 – 5). As additional controls, CreERT2 0/0/R26R+ littermates in both lines were injected with Tm (n = 3 – 4). Tissues were harvested 5 days – 8 weeks later; time to harvest was identical in experiments comparing the 2 SMC-Cre lines and similar results were obtained with R26R+/0 and R26R+/+ mice. As judged by X-Gal stain, after injection of Tm both alleles recombined R26R specifically and efficiently in both vascular and visceral SMC (Figure I in the online-only Data Supplement). Low-level SMC-specific Tm-independent “background” or “leaky” Cre activity (i.e., rare blue medial cells) was detected in vehicle-injected CreERT2 +/0 mice of both lines (Figure I in the online-only Data Supplement). High-level background SMC-specific Cre activity (i.e., numerous blue cells) was present in the muscular layer of the urinary bladder of CreERT2 +/0 R26R+ mice of both lines (Figure I in the online-only Data Supplement). Low-level background Cre activity was also detected by PCR of tail DNA (Figure II in the online-only Data Supplement). Despite this “leakiness,” expression from both SMC-Cre alleles appeared SMC-restricted both with and without Tm: no non-vascular blue cells were seen grossly or microscopically in SMC-poor tissues, including heart and skeletal muscle. There was no Cre activity (no blue cells) in Tm-injected SMC-CreERT2 0/0 R26R+ mice of either line.

To more precisely compare SMC-Cre activity in the two lines, we measured LacZ mRNA in the aortic media (see below) of Tm-treated Cre+/0 R26R+/+ mice of both lines. Expression of LacZ mRNA in the two lines was equivalent and far above background levels in Tm-injected Cre0/0 R26R+/+ controls (Figure III in the online-only Data Supplement). For the remainder of our experiments we compared Tm-treated Cre+/0 R26R+/+ Tgfbr2flox/flox mice with Tm-treated Cre0/0 R26R+/+ Tgfbr2flox/flox mice. This design compares Tgfbr2null/null SMC to Tgfbr2flox/flox SMC, eliminates concerns about background Cre activity, and controls for exposure to Tm, an agent that affects the vasculature directly.28–32

Measurement of TBRII Protein and Tgfbr2 mRNA in Aortic SMC

Because vascular Cre activity is limited to medial SMC (Figure I in the online-only Data Supplement)—and because our primary goal is to determine the role of Tgfbr2 in SMC—we focused our biochemical analyses on aortic medial tissue. We accomplished this by removing the aortic luminal endothelium, separating the media from the adventitia (Figure IV in the online-only Data Supplement), and extracting protein and RNA from the entire aortic media. Measurement of SMC markers in RNA extracted from either media or adventitia confirmed significant enrichment of SMC in medial tissue (Figure V in the online-only Data Supplement). Injection of Tm in Cre+/0 mice induced substantial reduction of TBRII protein in the aortic media of both lines (Figure 1). Surprisingly, however, when we measured Tgfbr2 mRNA using primers internal to the floxed Tgfbr2 exon 4 region, we found that aortic medial Tgfbr2 mRNA was either increased or unchanged in Tm-injected Cre+/0 mice of both lines (Figure VI in the online-only Data Supplement). To confirm that Cre had excised the expected region, we sequenced null-allele amplicons produced by PCR of tail DNA. The amplicons contained discontinuous sequences of Tgfbr2 separated by a loxP site (Figure II in the online-only Data Supplement), confirming Cre-mediated deletion of Tgfbr2. As an additional control we used exon 2-specific primers to measure Tgfbr2 mRNA. Again, we found increased Tgfbr2 mRNA after Tm injection, this time in Cre+/0 mice of both lines (Figure VI in the online-only Data Supplement). These results suggest compensatory increases in Tgfbr2 mRNA, but loss of TBRII protein in SMC of Tm-injected Cre+/0 mice.

Figure 1.

Figure 1

Open in a new tab

Knockdown of type II TGF-β receptor (TBRII) protein in aortic medial SMC. 4 weeks after treatment with tamoxifen, protein was extracted from the aortic media of Tgfbr2flox/flox mice that either lacked (Acta2-Cre0/0 and Myh11-Cre0/0) or expressed a CreERT2 transgene (Acta2-Cre+/0 and Myh11-Cre+/0). Western blots were probed with an antibody to TBRII or β-actin. Each lane is from a single mouse.

Loss of TBRII in Aortic SMC Disrupts Aortic Structure

Six-week-old Cre+/0 and Cre0/0 mice of both lines were injected with Tm daily for 5 days, killed 4 or 14 weeks after the last injection, and their aortas examined grossly and microscopically. Therefore, mice were either 11 weeks or 21 weeks of age when killed. At 4 weeks, gross aortic hemorrhage (defined as focal reddish-brown discoloration of the aortic wall) was present in ascending aorta (AscA)/aortic arch, descending thoracic aorta (DTA), or abdominal aorta (AA) of 32 of 84 (38%) Cre+/0 mice (19/58 Acta2-Cre+/0 and 13/26 Myh11-Cre+/0) and in none of the Cre0/0 mice (Figure 2 and Figure VII in the online-only Data Supplement). Hemorrhage was more common in the AscA and arch in both lines (31% of all Cre+/0 mice) than in the DTA or AA (7% in each area and only in the suprarenal AA; Table II). Sectioning of segments of AscA, DTA, and AA of randomly selected subsets of mice revealed intramural hematomas (IMH; defined as free red blood cells in the media; Figure 2 H, J, L) in 5 of 15 (33%) of Cre+/0 mice (both lines combined; results for each line are in Table II in the online only Data Supplement). Penetrating aortic ulcers (PAU; defined as a break in the intima and media exposing the inner media to the lumen; Figure 2 H and Figures VII H and VIII A and D in the online-only Data Supplement) were present in 14 of 15 (93%) of Cre+/0 mice almost exclusively in the AscA (both lines combined; results for each line are in Table II in the online only Data Supplement). Neither IMH nor PAU were present in any Cre0/0 mouse in either line (0 of 13; P = 0.04 and < 0.0001, respectively; all Cre+/0 versus all Cre0/0 mice). Rare, apparent aortic dissections (defined as an extended separation of medial layers containing a large pool of blood) were found in mice in this study and in a pilot group of mice not otherwise reported here (Figure 2 J and Figure VIII B and E in the online-only Data Supplement).

Figure 2.

Figure 2

Open in a new tab

Knockdown of Tgfbr2 in aortic SMC causes aortopathy. 4 weeks after treatment with tamoxifen, aortas were harvested from Tgfbr2flox/flox mice that either lacked (Acta2-Cre0/0; A, C, E, G, I, K) or expressed an Acta2-CreERT2 transgene (Acta2-Cre+/0; B, D, F, H, J, L). A – F, Representative pictures of ascending aorta (AscA) and arch, descending thoracic aorta (DTA), and abdominal aorta (AA). G – L, Representative transverse sections. H, Arrowhead: penetrating aortic ulcer. H, J, L, Arrows: aortic intramural hematoma or dissection (J). A – F, Ruler is in mm. G – L, Scale bar: 100 μm.

To determine whether aortic disease progressed over time we examined aortas from mice killed 14 weeks after Tm injection. At this time point, PAU were present in 13 of 14 (93%) Cre+/0 mice of both lines (almost exclusively in the AscA) and in 0/14 Cre0/0 mice (Table II; P < 0.0001). On gross examination, aortic discoloration suggestive of hemorrhage was noted in 10 of 18 Cre+/0 mice (56%) of both lines (most commonly in the AscA and arch; Table II) and in 0/14 Cre0/0 controls (P < 0.002). Surprisingly, microscopic examination of aortic sections of these mice (including all 10 aortas judged on gross examination to have aortic hemorrhage) revealed no free red blood cells in the aortic wall. To determine whether absence of red cells in aortic media of Cre+/0 mice 14 weeks after Tm injection was due to partial resolution of IMH, we stained AscA sections with Prussian blue. Sections of AscA from 9 of 14 (64%) of Cre+/0 mice of both lines stained with Prussian blue compared to 0 of 14 AscA of Cre0/0 controls (P = 0.0006; Table II and Figure VIII C and F in the online-only Data Supplement). Four weeks after Tm injection, Prussian blue staining was also present in 6 of 15 (40%) Cre+/0 mice of both lines and 0 of 13 controls (Table II; P < 0.02). Therefore, aortic hemorrhage begins shortly after deletion of Tgfbr2, resolves, and does not appear to recur.

Aortic wall morphology, evaluated on transverse sections, was abnormal in Cre+/0 mice of both lines. Four weeks after Tm injection, the lengths of the IEL and EEL of Cre+/0 aortas were increased marginally, if at all, compared to Cre0/0 controls of both lines. However, aortic medial thickness and medial cross-sectional area were increased in AscA, DTA, and AA of both lines (14 – 39% and 14 – 52%, respectively; Figure 3). All of these increases were significant for the Acta2-Cre line; increases of similar magnitude in the Myh11-Cre line were less uniformly significant, likely due to smaller group sizes (6 versus 7 – 9). Fourteen weeks after Tm injection, the IEL and EEL lengths of Cre+/0 aortas were significantly increased compared to Cre0/0 controls (6 – 32% and 6 – 31%, respectively; Figure 4), and lumens appeared larger (Figure 5 and Figure IX in the online-only Data Supplement). However, increased medial thickness in Cre+/0 versus Cre0/0 mice at 14 weeks (8 – 21%) was less pronounced than at 4 weeks, and was confined to the AscA and AA (Figure 4). In contrast, increases in medial area of Cre+/0 aortas were relatively larger at 14 versus 4 weeks (40 – 60% versus Cre0/0 controls) and were also confined to the AscA and AA (Figure 4). Therefore, SMC Tgfbr2 deletion causes early medial thickening followed by vessel dilation and relative medial thinning. That is, Cre+/0 aortas dilate between 4 and 14 weeks while their medial thickness changes relatively little (Cre+/0 medias thicken less from 4 – 14 weeks than Cre0/0 medias). Medial area in 14-week Cre+/0 aortas is increased primarily due to a larger circumference, not increased thickness. Overall, morphologic changes are far greater in the AscA and AA than the DTA, especially at 14 weeks. Abnormal aortic morphology (e.g., significantly increased IEL and EEL length) was present even in the most caudal AA sections, taken at the level of the left renal artery. The infrarenal AA was not analyzed histologically.

Figure 3.

Figure 3

Open in a new tab

Knockdown of Tgfbr2 in aortic SMC causes early medial thickening. 4 weeks after treatment with tamoxifen, aortas were harvested from Tgfbr2flox/flox mice that either lacked (Acta2-Cre0/0: open bars in A – D; and Myh11-Cre0/0: dark gray bars in E – H) or expressed a CreERT2 transgene (Acta2-Cre+/0: light gray bars in A – D; and Myh11-Cre+/0: black bars in E – H). Planimetry was performed on hematoxylin and eosin-stained sections from three segments: ascending aorta (AscA), descending thoracic aorta (DTA), and abdominal aorta (AA), with 5 step sections per segment per animal. A and E, Internal elastic lamina (IEL); B and F, external elastic lamina (EEL). *P < 0.05; **P < 0.01; ***P < 0.001 compared to Cre0/0 controls. n = 7 – 9 for Acta2-Cre groups; n = 6 for Myh11-Cre groups.

Figure 4.

Figure 4

Open in a new tab

Knockdown of Tgfbr2 in aortic SMC causes late aortic dilation. 14 weeks after treatment with tamoxifen, aortas were harvested from Tgfbr2flox/flox mice that either lacked (Acta2-Cre0/0: open bars in A – D; and Myh11-Cre0/0: dark gray bars in E – H) or expressed a CreERT2 transgene (Acta2-Cre+/0: light gray bars in A – D; and Myh11-Cre+/0: black bars in E – H). Planimetry was performed on hematoxylin and eosin-stained sections from three segments: ascending aorta (AscA), descending thoracic aorta (DTA), and abdominal aorta (AA), with 5 step sections per segment per animal. A and E, Internal elastic lamina (IEL); B and F, external elastic lamina (EEL). *P ≤ 0.05; **P < 0.01; ***P < 0.001 compared to Cre0/0 controls. n = 7 – 9 for Acta2-Cre groups; n = 6 for Myh11-Cre groups.

Figure 5.

Figure 5

Open in a new tab

Knockdown of Tgfbr2 in aortic SMC causes aortic dilation. 14 weeks after treatment with tamoxifen, aortas were harvested from Tgfbr2flox/flox mice that either lacked (Acta2-Cre0/0; A, C, and E) or expressed an Acta2-CreERT2 transgene (Acta2-Cre+/0; B, D, F). Representative sections of ascending aorta (AscA), descending thoracic aorta (DTA), and abdominal aorta (AA) are shown. B, Arrowheads: penetrating aortic ulcers. A – F, Scale bar: 200 μm.

Loss of TBRII in Aortic SMC Results in Elastin Damage, Increased Macrophage Markers, Cell Proliferation, and Matrix Accumulation

To further characterize aortic damage and potential healing responses after loss of SMC TBRII, we stained sections to detect elastin fibers, macrophage markers, cell proliferation, and extracellular matrix proteins. We measured elastin damage by counting elastic lamina breaks in AscA sections from both lines. Elastin breaks were 2 – 5-fold more frequent in Cre+/0 mice of both lines, at both time points (Figure X in the online-only Data Supplement; P < 0.01). In both lines, elastin breaks increased over time (P < 0.01).

We assessed the presence of macrophage markers by staining aortic sections for Mac-2. For both lines, Mac-2 staining was rare in sections from Cre0/0 mice but common in sections from Cre+/0 mice. The percentage of Mac-2-positive medial area was increased significantly in all three regions (3 – 8-fold; P < 0.05 except for 14-week DTA and AA in the Myh11-Cre line; Figure XI in the online-only Data Supplement). Mac-2 staining was typically located near an area of medial damage/hemorrhage, identified by presence of PAU, IMH, or Prussian blue stain on adjacent slides. An independent measure of macrophage marker accumulation, CD68 mRNA, was 4 – 5-fold more abundant in extracts from Cre+/0 versus Cre0/0 aortic media (P < 0.01 for both lines; Figure 6).

Figure 6.

Figure 6

Open in a new tab

Loss of aortic SMC Tgfbr2 alters expression of other genes. 4 weeks after treatment with tamoxifen, aortas were harvested from Tgfbr2flox/flox mice that either lacked (Acta2-Cre0/0: white bars in A; Myh11-Cre0/0: dark gray bars in B) or expressed a CreERT2 transgene (Acta2-Cre+/0: light gray bars in A; Myh11-Cre+/0: black bars in B). Gene expression was measured by qRT-PCR, using RNA isolated from the aortic media. *P < 0.05; **P < 0.01; ***P < 0.001 compared to Cre0/0 controls. n = 9 – 10 for Acta2-Cre groups; n = 6 for Myh11-Cre groups.

We measured cell proliferation in the AscA by pulse labeling with BrdU just before the 4-week time point. For both lines, BrdU-positive nuclei were rare in Cre0/0 aortas and significantly increased in Cre+/0 aortas (5 – 13-fold increases in total BrDU-positive nuclei; P ≤ 0.01). BrdU-positive cells were most common in the adventitia, with fewer along the luminal surface and even fewer in the media (Figure XII in the online-only Data Supplement).

Movat staining showed increased proteoglycan (PG) and collagen deposition in aortas of both lines of Cre+/0 mice at 4 and 14 weeks. PG and collagen accumulation were predominantly in the adventitia and most evident in AscA sections (Figure XIII in the online-only Data Supplement). An observer was blinded to genotype, given Movat-stained AscA sections from Cre+/0 and Cre0/0 mice of both lines, and asked to assign Cre genotype based only on extent of blue-green staining indicative of PG accumulation. The observer correctly genotyped 21 of 28 4-week mice and 27 of 28 14-week mice (P = 0.006 and < 0.0001, respectively).

We also stained sections from aortas of Acta2-Cre+/0 and Acta2-Cre0/0 mice for versican, a PG that is regulated by TGF-β33 and affects both SMC phenotype and tissue inflammation.34, 35 Versican staining was significantly increased in AscA and AA at both 4 and 14 weeks (4 – 47-fold, P < 0.001). Versican staining was typically highest in regions with PAU and adjacent Mac-2 staining (Figure XIV in the online-only Data Supplement).

Loss of TBRII Alters Aortic SMC Gene Expression

To begin to identify the mechanisms through which loss of SMC TBRII affects the aorta, 4 weeks after Tm treatment we isolated total aortic medial RNA and measured mRNA of several classes of genes: 1) genes involved in TGF-β signaling (Tgfbr1, Tgfbr3, Tgfb1, Tgfb2, Tgfb3, and Smad7); 2) genes involved in maintaining the differentiated SMC phenotype (Acta2, Myh11, Tagln, Smtn, and Cnn1); 3) Genes involved in extracellular matrix metabolism (Lox, Loxl1, Col1α1, Mmp2, Mmp9, and Mmp12); and 4) Canonical targets of TGF-β signaling: Ctgf and Serpine1; and 5) Other genes involved in aneurysm formation (Igf1, Ace).36

mRNA encoding Tgfbr3, Tgfb2, Tgfb3, and Smad7 were significantly increased after loss of TBRII in SMC from both lines, (2 – 4-fold; P ≤ 0.01 for all; Figure 6) Tgfb1 and Tgfbr1 mRNA were increased in Acta2-Cre+/0 mice (P < 0.03) and unchanged in Myh11-Cre+/0 mice. Genes encoding SMC lineage markers (Acta2, Myh11, Tagln, Smtn, and Cnn1 were upregulated in both lines (2 – 4-fold; P < 0.05 for all). Among genes involved in ECM metabolism, Loxl1 and Mmp2 were significantly upregulated in both lines (2 – 4-fold; P ≤ 0.005 and < 0.04, respectively). Lox, Col1α1, Mmp9, and Mmp12 were either unchanged or divergent between the lines. Ctgf mRNA decreased significantly in Acta2-Cre+/0 mice and trended lower (P = 0.06) in Myh11-Cre+/0 mice. Serpine1 mRNA trended lower in both lines (P = 0.1). Neither Igf1 nor Ace were significantly increased after loss of TBRII.

TBRII Knockdown in Cultured Aortic SMC Abrogates Canonical and Non-Canonical TGF-β Signaling

Aortic SMC from Acta2-Cre0/0Tgfbr2flox/flox mice were established in culture and treated with either AdCMVCre or AdCMVNull. Treatment with AdCMVCre reduced TBRII protein by 95% (P = 0.006; Figure 7 A and B). After treatment of SMC with either AdCMVCre or AdCMVNull, we added recombinant mouse TGF-β1 (1 ng/ml) to the cells. Cre-mediated deletion of Tgfbr2 significantly decreased TGF-β1 activation of both canonical (p-Smad2) and noncanonical (pERK and p-P38) TGF-β signaling pathways (60 – 90% reduction in peak levels; P < 0.05 for all; Figure 7 A, C – E).

Figure 7.

Figure 7

Open in a new tab

Knockdown of Tgfbr2 in cultured aortic SMC abrogates canonical and non-canonical TGF-β signaling. A, Aortic SMC from Acta2-Cre0/0 Tgfbr2flox/flox mice were transduced with either AdCMVNull or AdCMVCre. 24 hours later, cells were treated with mouse TGF-β1 (1 ng/ml) for 0 (no TGF-β1), 15, 60, and 180 minutes and cell extracts were analyzed by western blotting. B –E, Measurement of proteins by densitometry of western blots. TBRII signal was normalized to β-actin; p-Smad2 to Smad2, p-ERK1/2 to ERK1/2, and p-P38 to P38 in the same samples. *P < 0.05; **P < 0.01; ***P < 0.001 compared to AdCMVNull-treated cells by t-test (B) or 2-way ANOVA (C – E). Data in B – E are from 3 independent experiments, each as shown in A.

Loss of TBRII Alters Gene Expression in Cultured Aortic SMC

Cre-mediated loss of TBRII altered gene expression in cultured SMC in a pattern similar— although not identical—to that found in medial SMC of Cre+T mice (Figure XV in the online-only Data Supplement). mRNA encoding Tgfb1 was significantly increased (2-fold; P < 0.001), with trends towards increased expression of Tgfbr1, Tgfb2, and Tgfb3. mRNA encoding 3 of 4 SMC lineage markers were also increased (Myh11, Cnn1, and Tagln; 2 – 5-fold; P ≤ 0.01). Levels of Mmp2 and Mmp9 mRNA were far higher in cells lacking TBRII (8 – 10-fold; P ≤ 0.002).

Discussion

We used mice with inducible SMC-targeted Cre alleles and conditional Tgfbr2 alleles to delete TBRII in SMC and discover the consequences of loss of physiological SMC TGF-β signaling. Our major findings are: 1) Loss of TBRII in aortic SMC causes a major disruption of aortic wall structure within 4 weeks including intramural hemorrhage, wall thickening, ulceration, elastolysis, matrix accumulation, evidence of macrophage infiltration, and increased cell proliferation; 2) At a later time point (14 weeks) aortas that lack SMC TBRII are dilated with relative medial thinning, progressive elastolysis, and evidence of persistent macrophage accumulation; 3) Loss of TBRII in aortic SMC in vivo dramatically alters SMC expression of genes encoding proteins involved in TGF-β signaling, contractile function, and extracellular matrix metabolism; 4) Deletion of Tgfbr2 in cultured SMC rapidly alters expression of many of the same genes. Taken together, our results suggest that physiologic TGF-β signaling plays a critical role in maintaining aortic homeostasis and that loss of physiologic TGF-β signaling in SMC causes aortopathy that largely results from cell-autonomous effects on SMC.

We began this study by comparing 2 lines that express SMC-targeted inducible Cre recombinase.26, 27 Lines of transgenic mice can lose both transgene activity and tissue specificity over time;7, 37 therefore, we characterized both lines extensively. Both lines efficiently and specifically rearranged the R26R reporter allele in SMC, with no detectable differences between them. Both lines also produced essentially identical phenotypes when crossed into Tgfbr2flox/flox mice. Therefore, the theoretical concern that the Myh11-Cre allele must be used to avoid confounding Cre recombinase activity that could be present in non-SMC that express Acta238, 39 was not supported by our results. Others have also reported equivalent results with these 2 lines.40 Importantly, in this other study as well as ours, Cre activity was induced by Tm administration to mice without pre-existing vascular disease. The Acta2-Cre allele should probably be used cautiously in disease states because Acta2 is expressed by non-SMC in diseased vessels.38, 39 A new aspect of our study, as concerns characterization of these SMC-Cre alleles, is our finding of “background” (Tm-independent) Cre activity in both vascular and nonvascular SMC. This has not been reported by others27, 39 and should be accounted for in experimental designs, especially when clonal gene deletion could yield an important phenotype.41

Our results address the question of whether SMC TGF-β signaling is pathogenic or protective of aortic disease. This question is important because current clinical trials are based on the premise that TGF-β signaling is pathogenic.22, 23 If this premise is incorrect, these trials are unlikely to have positive outcomes. Arguments that TGF-β signaling in SMC is pathogenic are based on finding elevated pSMAD-2 in human aneurysmal tissue and in mouse models of human aortopathy;17, 20, 42 however, it is uncertain whether this finding is a cause or an effect of aortopathy,43 and pSMAD-2 can be increased via pathways independent of TGF-β.44 Moreover, elevated pSMAD-2 in diseased human aortas is not physically associated with TGF-β ligand accumulation, arguing that TGF-β is not the cause of pSMAD-2 upregulation.16 Proponents of the TGF-β-driven-aortopathy hypothesis bolster their arguments by pointing to a tissue “signature” of increased TGF-β signaling in diseased human and mouse aortas: elevated expression of connective tissue growth factor, collagen, and PAI-1.20, 42, 45 However, absence of this signature in diseased aortas is not accepted as evidence that TGF-β activity is not elevated, and therefore not pathogenic.19 Our results support a protective role for SMC TGF-β signaling by showing conclusively that physiologic SMC TGF-β signaling is required for postnatal aortic homeostasis and that disrupting SMC TGF-β signaling causes severe aortic pathology: intramural hematoma, penetrating aortic ulcer, dilation, and dissection. These results are consistent with other reports of salutary roles for TGF-β signaling in the aortic wall,14, 15, 24, 46 and decrease enthusiasm for human therapies that target aortic TGF-β signaling.

Our experiments were performed in young mice, raising concern that they may not apply to adult mice or humans.47 This concern is consistent with a recent report that systemic neutralization of TGF-β in Fbn1-deficient mice worsens aortopathy in young mice but prevents aortopathy in older mice.48 This report and the observation that deletion of SMC Tgfbr2 causes aortic dissection in younger but not older mice24 suggests that TGF-β inhibition may be dangerous in youth but protective later on.47 In a smaller study, however, we found serious aortic pathology (IMH, PAU) in a majority (70%) of mice (n = 10) in which SMC Tgfbr2 deletion was induced at 11 – 19 weeks of age (i.e., well into adult life; Figure VIII A and D and data not shown). This result (in which the prevalence of aortic pathology is likely underestimated because the aortas were sectioned less extensively than in the present study) suggests that inhibition of physiologic SMC TGF-β signaling may be unsafe at any age.

Our study does not identify the precise pathways through which loss of TBRII in aortic SMC causes aortopathy; however, it does provide some clues. Our in vitro studies show that SMC loss of TBRII causes rapid loss of both canonical and noncanonical TGF-β signaling pathways, and significant alterations in SMC gene expression. Analyses of aortic medial RNA reveal that altered SMC gene expression persists for at least 4 weeks after Tgfbr2 deletion and is accompanied by gross and microscopic evidence of medial and endothelial damage: loss of endothelial barrier function leading to entry of blood into the media, loss of medial cell cohesiveness leading to ulceration and dissection, and medial elastolysis. Loss of barrier function, medial cell cohesiveness, and elastolysis are all plausible sequelae of the alterations in SMC gene expression that we describe: increased MMP expression and dysregulation of SMC contractile proteins. Medial thickening at 4 weeks could be due to SMC hypertrophy, edema due to increased permeability, macrophage infiltration, IMH that have partially resolved, or a combination of these processes. Because cell proliferation affects only a small minority of medial cells, medial cell proliferation appears to play a minor role. Aortic dilation and PAU 14 weeks after SMC loss of TBRII are likely due to persistent medial SMC dysfunction. Increased Mac-2 abundance throughout the vessel wall and enhanced matrix deposition and cell proliferation in the adventitia likely reflect compensatory healing responses to medial SMC dysfunction. According to this model (Figure XVI in the online-only Data Supplement) aortopathy in mice with SMC-specific loss of TBRII is driven by cell-autonomous events in medial SMC. This model is supported by our in vitro data showing significant changes in aortic SMC gene expression after loss of TBRII, in the absence of exposure to other cell types.

In both the in vitro and in vivo studies presented here, loss of SMC TBRII caused upregulation of SMC expression of TGF-β signaling components: ligands, receptors, and a regulator of canonical TGF-β signaling (Smad7). These findings raise the possibility that compensatory, cell-autonomous upregulation of TGF-β signaling occurs after loss of TBRII, as reported in murine palatal mesenchymal cells with Tgfbr2 deletion.49 Upregulation of TGF-β signaling in TBRII-null SMC—if present—would be congruent with reports of “paradoxical” upregulation of TGF-β signaling in humans with loss-of-function mutations in TGF-β signaling components.20, 42, 50 This paradoxical upregulation of TGF-β signaling is thought to drive these human aortopathies and could potentially explain aortopathy in the present study. SMC upregulation of Myh11, Tagln, and Acta2 mRNA would be consistent with increased TGF-β signaling. However, our data are inconsistent with simple upregulation of either canonical or noncanonical TGF-β signaling. In vitro, TBRII-null SMC appear to lose both canonical and noncanonical signaling in response to TGF-β ligand. In vivo, we found no evidence that loss of TBRII leads to upregulated expression of well-established TGF-β-responsive genes (Col1α1, Ctgf, and Serpine1). In summary, although TGF-β signaling components are upregulated after SMC-specific loss of TBRII, it is unclear whether SMC TGF-β signaling increases, and if so, whether this drives the aortopathy or is an epiphenomenon.

While this study was in progress, another group reported use of the Myh11-CreERT2 allele to delete a different Tgfbr2 exon (exon 2) in postnatal mice.24 Both studies found that SMC loss of TBRII in young mice causes aortopathy with early medial thickening, elastin fragmentation, adventitial fibrosis and proliferation, as well as later medial atrophy and aortic dilation. Both studies also found elevated Tgfb2 and Tgfb3 mRNA after Tgfbr2 deletion in vivo as well as loss of both canonical and noncanonical TGF-β signaling in vitro in SMC lacking TBRII. Our study adds new information by including a more detailed histological characterization of the aortopathy (including a small study in older mice), by characterizing aberrant SMC gene expression that occurs early after loss of TBRII (in vitro), by focusing our in vivo gene expression studies on aortic media, and by including females (approximately 50% of Acta2-Cre+/0 mice). Our novel findings include: 1) the aortopathy includes the abdominal aorta and is least pronounced in the descending thoracic aorta (regional variability of aortic pathology could potentially be due to variations in the embryonic origin of SMC, with ascending aortic SMC derived from neural crest, descending thoracic aortic SMC from somites, and abdominal aortic SMC from mesoderm);51 2) aortopathy is common after SMC Tgfbr2 deletion in older mice; 3) mRNA encoding SMC contractile proteins are upregulated in the aortic media (and in cultured SMC) after Tgfbr2 deletion whereas Col1α1 is not upregulated; 4) evidence of leukocytic infiltration is not limited to the adventitia; 5) cell proliferation after loss of SMC Tgfbr2 occurs predominantly in the adventitia; and 6) aortic hematomas and dissections appear confined to the early period after Tgfbr2 deletion and resolve over time. Moreover, our in vitro gene expression data suggest that SMC dysfunction and upregulated MMP expression occur early after SMC loss of TBRII. When combined with the modest effect of TBRII loss on medial proliferation, our data support SMC dysfunction as a primary cause of aortopathy after loss of TBRII, and tend to discount a critical role for adventitial growth factor-driven proliferative medial disease.24

Loss-of-function mutations in TGFBR2 cause aortopathy in humans,42, 52 raising the question of whether our data provide mechanistic insight into this human disease. We believe our data do provide insight, but with a caveat. Specifically, our finding that disruption of SMC TGF-β signaling causes SMC dysfunction and aortopathy identifies SMC dysfunction as a likely cause of aortic disease in humans with TGFBR2 mutations. However, we are hesitant to derive mechanistic insights beyond this, because the TGFBR2 mutants that cause human aortic disease are not null alleles42, 52, 53 and it is uncertain to what extent acute homozygous receptor knockout can model a disease that is caused by the lifelong presence of heterozygous mutant receptors. The most important application of this study to human health is the unambiguous lesson that physiologic TGF-β signaling is vital for postnatal aortic health. Strategies aimed at blocking TGF-β signaling in humans should take this into account.

Supplementary Material

Methods and Material
Supplementary data

Significance.

Intracellular signals initiated by TGF-β ligands and transduced by the type II TGF-β receptor in SMC play essential roles in SMC differentiation and vascular development. However, the contribution of these signals to postnatal aortic homeostasis is less well defined. By deleting the type II TGF-β receptor specifically in SMC of adult mice, we determined that SMC TGF-β signaling plays a critical role in maintaining postnatal aortic health. Specifically, mice that lack physiologic SMC TGF-β signaling develop severe aortic pathology including hemorrhage, ulceration, elastolysis, and aneurysmal dilation. Aortic pathology extends along the full length of the aorta and worsens over time. These results are clinically relevant because several human aortopathies are currently attributed to excess SMC TGF-β signaling and therapies that aim to block SMC TGF-β signaling are proposed for human trials. Our results suggest that blocking SMC TGF-β signaling may worsen—not prevent—aortic disease.

Acknowledgments

We thank Dr. Jude Alsarraj for technical assistance, members of the laboratory of Dr. Cecilia Giachelli for teaching us the aortic medial isolation protocol, Dr. Pamela Johnson at the Histology Core of the Benaroya Research Institute for performing Movat’s staining and versican immunostaining, Dr. Stefan Offermanns for the Myh11-CreERT2 mice (originally termed SMMHC-CreERT2) and Drs. Daniel Metzger and Pierre Chambon for the Acta2-CreERT2 mice (originally termed SMA-CreERT2).

Source of Funding

This study was supported by grants (to D.A.D.) from the American Heart Association, the National Heart, Lung, and Blood Institute (HL116612) and the John L. Locke Jr. Charitable Trust and by grants to T.N.W. from the N.I.H. (EB012558 and HL098067). S.A. was supported by T32HL007828.

Abbreviations

AA

abdominal aorta

AscA

ascending aorta

DTA

descending thoracic aorta

IMH

intramural hematoma

PAU

penetrating aortic ulcer

PG

proteoglycan

TBRII

type II transforming growth factor-beta receptor

TGF-β

transforming growth factor beta

Tm

tamoxifen

Footnotes

Disclosures

The authors declare no conflicts.

References

  • 1.Bjorkerud S. Effects of transforming growth factor-beta 1 on human arterial smooth muscle cells in vitro. Arterioscler Thromb. 1991;11:892–902. [PubMed] [Google Scholar]
  • 2.Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004;84:767–801. doi: 10.1152/physrev.00041.2003. [DOI] [PubMed] [Google Scholar]
  • 3.Saltis J, Agrotis A, Bobik A. Regulation and interactions of transforming growth factor-b with cardiovascular cells: Implications for development and disease. Clin Exp Pharmacol Physiol. 1996;23:193–200. doi: 10.1111/j.1440-1681.1996.tb02595.x. [DOI] [PubMed] [Google Scholar]
  • 4.Goumans MJ, Liu Z, ten Dijke P. Tgf-beta signaling in vascular biology and dysfunction. Cell Res. 2009;19:116–127. doi: 10.1038/cr.2008.326. [DOI] [PubMed] [Google Scholar]
  • 5.Sinha S, Hoofnagle MH, Kingston PA, McCanna ME, Owens GK. Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells. Am J Physiol Cell Physiol. 2004;287:C1560–1568. doi: 10.1152/ajpcell.00221.2004. [DOI] [PubMed] [Google Scholar]
  • 6.Davis-Dusenbery BN, Chan MC, Reno KE, Weisman AS, Layne MD, Lagna G, Hata A. Downregulation of klf4 by mir-143/145 is critical for modulation of vascular smooth muscle cell phenotype by tgf-{beta} and bmp. J Biol Chem. 2011;286:28097–28110. doi: 10.1074/jbc.M111.236950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Frutkin AD, Shi H, Otsuka G, Leveen P, Karlsson S, Dichek DA. A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing sm22-cre, not smmhc-cre. J Mol Cell Cardiol. 2006;41:724–731. doi: 10.1016/j.yjmcc.2006.06.067. [DOI] [PubMed] [Google Scholar]
  • 8.Carvalho RL, Itoh F, Goumans MJ, Lebrin F, Kato M, Takahashi S, Ema M, Itoh S, van Rooijen M, Bertolino P, Ten Dijke P, Mummery CL. Compensatory signalling induced in the yolk sac vasculature by deletion of tgfbeta receptors in mice. J Cell Sci. 2007;120:4269–4277. doi: 10.1242/jcs.013169. [DOI] [PubMed] [Google Scholar]
  • 9.Langlois D, Hneino M, Bouazza L, Parlakian A, Sasaki T, Bricca G, Li JY. Conditional inactivation of tgf-beta type ii receptor in smooth muscle cells and epicardium causes lethal aortic and cardiac defects. Transgenic Res. 2010;19:1069–1082. doi: 10.1007/s11248-010-9379-4. [DOI] [PubMed] [Google Scholar]
  • 10.Jaffe M, Sesti C, Washington I, Du L, Dronadula N, Chin MT, Stolz DB, Davis EC, Dichek DA. Transforming growth factor beta signaling in myogenic cells regulates vascular morphogenesis, differentiation, and matrix synthesis. Arterioscler Thromb Vasc Biol. 2012;32:e1–e11. doi: 10.1161/ATVBAHA.111.238410. [DOI] [PubMed] [Google Scholar]
  • 11.Schulick AH, Taylor AJ, Zuo W, Qiu C-B, Dong G, Woodward RN, Agah R, Roberts AB, Virmani R, Dichek DA. Overexpression of transforming growth factor b1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia. Proc Natl Acad Sci U S A. 1998;95:6983–6988. doi: 10.1073/pnas.95.12.6983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Nabel EG, Shum L, Pompili VJ, Yang Z-Y, San H, Shu HB, Liptay S, Gold L, Gordon D, Derynck R, Nabel GJ. Direct transfer of transforming growth factor b1 gene into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci U S A. 1993;90:10759–10763. doi: 10.1073/pnas.90.22.10759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Otsuka G, Agah R, Frutkin AD, Wight TN, Dichek DA. Transforming growth factor beta 1 induces neointima formation through plasminogen activator inhibitor-1-dependent pathways. Arterioscler Thromb Vasc Biol. 2006;26:737–743. doi: 10.1161/01.ATV.0000201087.23877.e1. [DOI] [PubMed] [Google Scholar]
  • 14.Dai J, Losy F, Guinault AM, Pages C, Anegon I, Desgranges P, Becquemin JP, Allaire E. Overexpression of transforming growth factor-beta1 stabilizes already-formed aortic aneurysms: A first approach to induction of functional healing by endovascular gene therapy. Circulation. 2005;112:1008–1015. doi: 10.1161/CIRCULATIONAHA.104.523357. [DOI] [PubMed] [Google Scholar]
  • 15.Wang Y, Ait-Oufella H, Herbin O, Bonnin P, Ramkhelawon B, Taleb S, Huang J, Offenstadt G, Combadiere C, Renia L, Johnson JL, Tharaux PL, Tedgui A, Mallat Z. Tgf-beta activity protects against inflammatory aortic aneurysm progression and complications in angiotensin ii-infused mice. J Clin Invest. 2010;120:422–432. doi: 10.1172/JCI38136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Gomez D, Al Haj Zen A, Borges LF, Philippe M, Gutierrez PS, Jondeau G, Michel JB, Vranckx R. Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the smad2 pathway. J Pathol. 2009;218:131–142. doi: 10.1002/path.2516. [DOI] [PubMed] [Google Scholar]
  • 17.Habashi JP, Judge DP, Holm TM, et al. Losartan, an at1 antagonist, prevents aortic aneurysm in a mouse model of marfan syndrome. Science. 2006;312:117–121. doi: 10.1126/science.1124287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Gallo EM, Loch DC, Habashi JP, et al. Angiotensin ii-dependent tgf-beta signaling contributes to loeys-dietz syndrome vascular pathogenesis. J Clin Invest. 2014;124:448–460. doi: 10.1172/JCI69666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Doyle AJ, Doyle JJ, Bessling SL, et al. Mutations in the tgf-beta repressor ski cause shprintzen-goldberg syndrome with aortic aneurysm. Nat Genet. 2012;44:1249–1254. doi: 10.1038/ng.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lindsay ME, Schepers D, Bolar NA, et al. Loss-of-function mutations in tgfb2 cause a syndromic presentation of thoracic aortic aneurysm. Nat Genet. 2012;44:922–927. doi: 10.1038/ng.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Lacro RV, Dietz HC, Wruck LM, et al. Rationale and design of a randomized clinical trial of beta-blocker therapy (atenolol) versus angiotensin ii receptor blocker therapy (losartan) in individuals with marfan syndrome. Am Heart J. 2007;154:624–631. doi: 10.1016/j.ahj.2007.06.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Brooke BS, Habashi JP, Judge DP, Patel N, Loeys B, Dietz HC., 3rd Angiotensin ii blockade and aortic-root dilation in marfan’s syndrome. N Engl J Med. 2008;358:2787–2795. doi: 10.1056/NEJMoa0706585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lacro RV, Dietz HC, Sleeper LA, et al. Atenolol versus losartan in children and young adults with marfan’s syndrome. N Engl J Med. 2014;371:2061–2071. doi: 10.1056/NEJMoa1404731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Li W, Li Q, Jiao Y, Qin L, Ali R, Zhou J, Ferruzzi J, Kim RW, Geirsson A, Dietz HC, Offermanns S, Humphrey JD, Tellides G. Tgfbr2 disruption in postnatal smooth muscle impairs aortic wall homeostasis. J Clin Invest. 2014;124:755–767. doi: 10.1172/JCI69942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Leveen P, Larsson J, Ehinger M, Cilio CM, Sundler M, Sjostrand LJ, Holmdahl R, Karlsson S. Induced disruption of the transforming growth factor beta type ii receptor gene in mice causes a lethal inflammatory disorder that is transplantable. Blood. 2002;100:560–568. doi: 10.1182/blood.v100.2.560. [DOI] [PubMed] [Google Scholar]
  • 26.Wendling O, Bornert JM, Chambon P, Metzger D. Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse. Genesis. 2009;47:14–18. doi: 10.1002/dvg.20448. [DOI] [PubMed] [Google Scholar]
  • 27.Wirth A, Benyo Z, Lukasova M, Leutgeb B, Wettschureck N, Gorbey S, Orsy P, Horvath B, Maser-Gluth C, Greiner E, Lemmer B, Schutz G, Gutkind JS, Offermanns S. G12-g13-larg-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat Med. 2008;14:64–68. doi: 10.1038/nm1666. [DOI] [PubMed] [Google Scholar]
  • 28.Perez GJ. Dual effect of tamoxifen on arterial kca channels does not depend on the presence of the beta1 subunit. J Biol Chem. 2005;280:21739–21747. doi: 10.1074/jbc.M413953200. [DOI] [PubMed] [Google Scholar]
  • 29.Leung HS, Yung LM, Leung FP, Yao X, Chen ZY, Ko WH, Laher I, Huang Y. Tamoxifen dilates porcine coronary arteries: Roles for nitric oxide and ouabain-sensitive mechanisms. Br J Pharmacol. 2006;149:703–711. doi: 10.1038/sj.bjp.0706921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ning N, Lin G, Lue TF, Lin CS. Effects of estrogen, raloxifene, and levormeloxifene on the expression of rho-kinase signaling molecules in urethral smooth muscle cells. Urology. 2010;76:1517e1516–1511. doi: 10.1016/j.urology.2010.07.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Delle H, Rocha JR, Cavaglieri RC, Vieira JM, Jr, Malheiros DM, Noronha IL. Antifibrotic effect of tamoxifen in a model of progressive renal disease. J Am Soc Nephrol. 2012;23:37–48. doi: 10.1681/ASN.2011010046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Savolainen-Peltonen H, Luoto NM, Kangas L, Hayry P. Selective estrogen receptor modulators prevent neointima formation after vascular injury. Mol Cell Endocrinol. 2004;227:9–20. doi: 10.1016/j.mce.2004.08.004. [DOI] [PubMed] [Google Scholar]
  • 33.Schönherr E, Järveläinen HT, Sandell LJ, Wight TN. Effects of platelet-derived growth factor and transforming growth factor-b1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells. J Biol Chem. 1991;266:17640–17647. [PubMed] [Google Scholar]
  • 34.Wight TN, Kang I, Merrilees MJ. Versican and the control of inflammation. Matrix Biol. 2014;35:152–161. doi: 10.1016/j.matbio.2014.01.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Kang I, Barth JL, Sproul EP, Yoon DW, Braun KR, Argraves WS, Wight TN. Expression of v3 versican by rat arterial smooth muscle cells promotes differentiated and anti-inflammatory phenotypes. J Biol Chem. 2015;290:21629–21641. doi: 10.1074/jbc.M115.657486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Pannu H, Tran-Fadulu V, Papke CL, Scherer S, Liu Y, Presley C, Guo D, Estrera AL, Safi HJ, Brasier AR, Vick GW, Marian AJ, Raman CS, Buja LM, Milewicz DM. Myh11 mutations result in a distinct vascular pathology driven by insulin-like growth factor 1 and angiotensin ii. Human molecular genetics. 2007;16:2453–2462. doi: 10.1093/hmg/ddm201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Lee S, Agah R, Xiao M, Frutkin AD, Kremen M, Shi H, Dichek DA. Regulated transgene expression in vascular smooth muscle (letter) Circ Res. 2005;97:85. [PubMed] [Google Scholar]
  • 38.Gomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc Res. 2012;95:156–164. doi: 10.1093/cvr/cvs115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Shankman LS, Gomez D, Cherepanova OA, Salmon M, Alencar GF, Haskins RM, Swiatlowska P, Newman AA, Greene ES, Straub AC, Isakson B, Randolph GJ, Owens GK. Klf4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. Nat Med. 2015;21:628–637. doi: 10.1038/nm.3866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Herring BP, Hoggatt AM, Burlak C, Offermanns S. Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury. Vasc Cell. 2014;6:21. doi: 10.1186/2045-824X-6-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.McCaffrey TA, Du B, Consigli S, Szabo P, Bray PJ, Hartner L, Weksler BB, Sanborn TA, Bergman G, Bush HL., Jr Genomic instability in the type ii tgf-b1 receptor gene in atherosclerotic and restenotic vascular cells. J Clin Invest. 1997;100:2182–2188. doi: 10.1172/JCI119754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Loeys BL, Chen J, Neptune ER, et al. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in tgfbr1 or tgfbr2. Nat Genet. 2005;37:275–281. doi: 10.1038/ng1511. [DOI] [PubMed] [Google Scholar]
  • 43.Gomez D, Coyet A, Ollivier V, Jeunemaitre X, Jondeau G, Michel JB, Vranckx R. Epigenetic control of vascular smooth muscle cells in marfan and non-marfan thoracic aortic aneurysms. Cardiovasc Res. 2011;89:446–456. doi: 10.1093/cvr/cvq291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Akhurst RJ. The paradoxical tgf-beta vasculopathies. Nat Genet. 2012;44:838–839. doi: 10.1038/ng.2366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Lindsay ME, Dietz HC. Lessons on the pathogenesis of aneurysm from heritable conditions. Nature. 2011;473:308–316. doi: 10.1038/nature10145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Frutkin AD, Otsuka G, Stempien-Otero A, Sesti C, Du L, Jaffe M, Dichek HL, Pennington CJ, Edwards DR, Nieves-Cintron M, Minter D, Preusch M, Hu JH, Marie JC, Dichek DA. Tgf-beta1 limits plaque growth, stabilizes plaque structure, and prevents aortic dilation in apolipoprotein e-null mice. Arterioscler Thromb Vasc Biol. 2009;29:1251–1257. doi: 10.1161/ATVBAHA.109.186593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Lacro RV, Dietz HC, Mahony L. Atenolol versus losartan in marfan’s syndrome. N Engl J Med. 2015;372:980–981. doi: 10.1056/NEJMc1500128. [DOI] [PubMed] [Google Scholar]
  • 48.Cook JR, Clayton NP, Carta L, Galatioto J, Chiu E, Smaldone S, Nelson CA, Cheng SH, Wentworth BM, Ramirez F. Dimorphic effects of transforming growth factor-beta signaling during aortic aneurysm progression in mice suggest a combinatorial therapy for marfan syndrome. Arterioscler Thromb Vasc Biol. 2015;35:911–917. doi: 10.1161/ATVBAHA.114.305150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Iwata J, Hacia JG, Suzuki A, Sanchez-Lara PA, Urata M, Chai Y. Modulation of noncanonical tgf-beta signaling prevents cleft palate in tgfbr2 mutant mice. J Clin Invest. 2012;122:873–885. doi: 10.1172/JCI61498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Boileau C, Guo DC, Hanna N, et al. Tgfb2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of marfan syndrome. Nat Genet. 2012;44:916–922. doi: 10.1038/ng.2348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Majesky MW. Developmental basis of vascular smooth muscle diversity. Arterioscler Thromb Vasc Biol. 2007;27:1248–1258. doi: 10.1161/ATVBAHA.107.141069. [DOI] [PubMed] [Google Scholar]
  • 52.Mizuguchi T, Collod-Beroud G, Akiyama T, et al. Heterozygous tgfbr2 mutations in marfan syndrome. Nat Genet. 2004;36:855–860. doi: 10.1038/ng1392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Horbelt D, Guo G, Robinson PN, Knaus P. Quantitative analysis of tgfbr2 mutations in marfan-syndrome-related disorders suggests a correlation between phenotypic severity and smad signaling activity. J Cell Sci. 2010;123:4340–4050. doi: 10.1242/jcs.074773. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Methods and Material
NIHMS730872-supplement-Methods_and_Material.pdf (173.6KB, pdf)
Supplementary data
NIHMS730872-supplement-Supplementary_data.pdf (3.4MB, pdf)

RESOURCES