Mark E. Lindsay

Noncanonical TGFβ signaling contributes to aortic aneurysm progression in Marfan syndrome mice.

Transforming growth factor–β promotes aortic aneurysm formation through activation of its “noncanonical” signaling pathway. Transforming growth factor–β (TGFβ) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFβ can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal–regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFβ. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase–1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFβ signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.

Lessons on the pathogenesis of aneurysm from heritable conditions

Aortic aneurysm is common, accounting for 1–2% of all deaths in industrialized countries. Early theories of the causes of human aneurysm mostly focused on inherited or acquired defects in components of the extracellular matrix in the aorta. Although several mutations in the genes encoding extracellular matrix proteins have been recognized, more recent discoveries have shown important perturbations in cytokine signalling cascades and intracellular components of the smooth muscle contractile apparatus. The modelling of single-gene heritable aneurysm disorders in mice has shown unexpected involvement of the transforming growth factor-β cytokine pathway in aortic aneurysm, highlighting the potential for new therapeutic strategies.

Loss-of-function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm

Loeys-Dietz syndrome (LDS) associates with a tissue signature for high transforming growth factor (TGF)-β signaling but is often caused by heterozygous mutations in genes encoding positive effectors of TGF-β signaling, including either subunit of the TGF-β receptor or SMAD3, thereby engendering controversy regarding the mechanism of disease. Here, we report heterozygous mutations or deletions in the gene encoding the TGF-β2 ligand for a phenotype within the LDS spectrum and show upregulation of TGF-β signaling in aortic tissue from affected individuals. Furthermore, haploinsufficient Tgfb2+/− mice have aortic root aneurysm and biochemical evidence of increased canonical and noncanonical TGF-β signaling. Mice that harbor both a mutant Marfan syndrome (MFS) allele (Fbn1C1039G/+) and Tgfb2 haploinsufficiency show increased TGF-β signaling and phenotypic worsening in association with normalization of TGF-β2 expression and high expression of TGF-β1. Taken together, these data support the hypothesis that compensatory autocrine and/or paracrine events contribute to the pathogenesis of TGF-β–mediated vasculopathies.

Regeneration Next: Toward Heart Stem Cell Therapeutics

Stem cell biology holds great promise for a new era of cell-based therapy, sparking considerable interest among scientists, clinicians, and their patients. However, the translational arm of stem cell science is in a relatively primitive state. Although a number of clinical studies have been initiated, the early returns point to several inherent problems. In this regard, the clinical potential of stem cells can only be fully realized by the identification of the key barriers to clinical implementation. Here, we examine experimental paradigms to address the critical steps in the transition from stem cell biology to regenerative medicine, utilizing cardiovascular disease as a case study.

Mutations in the TGF-β Repressor SKI Cause Shprintzen-Goldberg Syndrome with Aortic Aneurysm

Elevated transforming growth factor (TGF)-β signaling has been implicated in the pathogenesis of syndromic presentations of aortic aneurysm, including Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS). However, the location and character of many of the causal mutations in LDS intuitively imply diminished TGF-β signaling. Taken together, these data have engendered controversy regarding the specific role of TGF-β in disease pathogenesis. Shprintzen-Goldberg syndrome (SGS) has considerable phenotypic overlap with MFS and LDS, including aortic aneurysm. We identified causative variation in ten individuals with SGS in the proto-oncogene SKI, a known repressor of TGF-β activity. Cultured dermal fibroblasts from affected individuals showed enhanced activation of TGF-β signaling cascades and higher expression of TGF-β–responsive genes relative to control cells. Morpholino-induced silencing of SKI paralogs in zebrafish recapitulated abnormalities seen in humans with SGS. These data support the conclusions that increased TGF-β signaling is the mechanism underlying SGS and that high signaling contributes to multiple syndromic presentations of aortic aneurysm.

Angiotensin II–dependent TGF-β signaling contributes to Loeys-Dietz syndrome vascular pathogenesis

Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysm and dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. LDS is caused by heterozygous missense mutations in either TGF-β receptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-β signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-β signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-β in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-β signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-β target gene output, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-β1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-β1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-β signaling contributes to postnatal aneurysm progression in LDS.

Npap60/Nup50 Is a Tri-Stable Switch that Stimulates Importin-α:β-Mediated Nuclear Protein Import

Abstract Many nuclear-targeted proteins are transported through the nuclear pore complex (NPC) by the importin-α:β receptor. We now show that Npap60 (also called Nup50), a protein previously believed to be a structural component of the NPC, is a Ran binding protein and a cofactor for importin-α:β-mediated import. Npap60 is a tri-stable switch that alternates between binding modes. The C terminus binds importin-β through RanGTP. The N terminus binds the C terminus of importin-α, while a central domain binds importin-β. Npap60:importin-α:β binds cargo and can stimulate nuclear import. Endogenous Npap60 can shuttle and is accessible from the cytoplasmic side of the nuclear envelope. These results identify Npap60 as a cofactor for importin-α:β nuclear import and as a previously unidentified subunit of the importin complex.

The Genetic Basis of Aortic Aneurysm

Gene identification in human aortic aneurysm conditions is proceeding at a rapid pace and the integration of pathogenesis-based management strategies in clinical practice is an emerging reality. Human genetic alterations causing aneurysm involve diverse gene products including constituents of the extracellular matrix, cell surface receptors, intracellular signaling molecules, and elements of the contractile cytoskeleton. Animal modeling experiments and human genetic discoveries have extensively implicated the transforming growth factor-β (TGF-β) cytokine-signaling cascade in aneurysm progression, but mechanistic links between many gene products remain obscure. This chapter will integrate human genetic alterations associated with aortic aneurysm with current basic research findings in an attempt to form a reconciling if not unifying model for hereditary aortic aneurysm.

Hereditary Influence in Thoracic Aortic Aneurysm and Dissection

Thoracic aortic aneurysm is a potentially life-threatening condition in that it places patients at risk for aortic dissection or rupture. However, our modern understanding of the pathogenesis of thoracic aortic aneurysm is quite limited. A genetic predisposition to thoracic aortic aneurysm has been established, and gene discovery in affected families has identified several major categories of gene alterations. The first involves mutations in genes encoding various components of the transforming growth factor beta (TGF-β) signaling cascade (FBN1, TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, SMAD3 and SKI), and these conditions are known collectively as the TGF-β vasculopathies. The second set of genes encode components of the smooth muscle contractile apparatus (ACTA2, MYH11, MYLK, and PRKG1), a group called the smooth muscle contraction vasculopathies. Mechanistic hypotheses based on these discoveries have shaped rational therapies, some of which are under clinical evaluation. This review discusses published data on genes involved in thoracic aortic aneurysm and attempts to explain divergent hypotheses of aneurysm origin.

Risk of rupture or dissection in descending thoracic aortic aneurysm.

Background— Current practice guidelines recommend surgical repair of large thoracic aortic aneurysms to prevent fatal aortic dissection or rupture, but limited natural history data exist to support clinical criteria for timely intervention. Methods and Results— Of 3247 patients with thoracic aortic aneurysm registered in our institutional Thoracic Aortic Center Database, we identified and reviewed 257 nonsyndromic patients (age, 72.4±10.5 years; 143 female) with descending thoracic or thoracoabdominal aortic aneurysm without a history of aortic dissection in whom surgical intervention was not undertaken. The primary end point was a composite of aortic dissection/rupture and sudden death. Baseline mean maximal aortic diameter was 52.4±10.8 mm, with 103 patients having diameters ≥55 mm. During a median follow-up of 25.1 months (quartiles 1–3, 8.3–56.4 months), definite and possible aortic events occurred in 19 (7.4%) and 31 (12.1%) patients, respectively. On multivariable analyses, maximal aortic diameter at baseline emerged as the only significant predictor of aortic events (hazard ratio=1.12; 95% confidence interval, 1.08–1.15). Estimated rates of definite aortic events within 1 year were 5.5%, 7.2%, and 9.3% for aortic diameters of 50, 55, and 60 mm, respectively. Receiver-operating characteristic curves for discriminating aortic events were higher for indexed aortic sizes referenced by body size (area under the curve=0.832–0.889) but not significantly different from absolute maximal aortic diameter (area under the curve=0.805). Conclusions— Aortic size was the principal factor related to aortic events in unrepaired descending thoracic or thoracoabdominal aortic aneurysm. Although the risk of aortic events started to increase with a diameter >5.0 to 5.5 cm, it is uncertain whether repair of thoracic aortic aneurysms in this range leads to overall benefit, and the threshold for repair requires further evaluation.