Prior to 2003, pathogenic models for Marfan syndrome (MFS - and other disorders caused by deficiency of a structural matrix protein) singularly invoked inherent weakness of the tissues as the driving force in disease progression. This suggested that children with these conditions are born with an obligate structural predisposition for the tissues to fail later in life and boded poorly for the development of productive postnatal treatments. Dietz was the first to recognize that many of the clinical manifestations of MFS – including low muscle mass and fat stores or craniofacial dysmorphism – were difficult or impossible to reconcile with this “weak tissues” model. A collaborative team that includes the Dietz, Ramirez, Sakai and Rifkin labs went on to show that fibrillin-1 (the deficient gene product in MFS) serves a second and important regulatory role by sequestering the large latent complex of the growth factor TGFbeta and suppressing its activation and signaling. Using developmental emphysema as a read-out, Enid Nepture showed that suppression of TGFbeta activity using a neutralizing antibody could greatly attenuate or even prevent disease in a fibrillin-1 deficient mouse model of MFS. Later work in the Dietz lab showed that the protection afforded by TGFbeta antagonism extended to other phenotypes in Marfan mice including congenital mitral valve prolapse with mxyomatous valve degeneration. The relevance of this mechanism to manifestations that more intuitively reflect failed tissue homeostasis (e.g. aneurysm) was unknown. Using fibrillin-1 deficient mice, Dietz demonstrated increased TGFbeta signaling (nuclear pSmad2) and output of TGFbeta-responsive genes in the aortic wall. Systemic delivery of a TGFbeta-neutralizing antibody attenuated aortic root enlargement and improved aortic wall architecture. In an attempt to become more clinically relevant, the Dietz lab turned their attention to losartan, an FDA-approved angiotensin II type 1 (AT1) receptor blocker that both lowers blood pressure and had been shown to inhibit TGFbeta signaling in animal models of chronic renal disease. A long-term trial showed that losartan fully normalizes TGFbeta signaling, aortic root growth and size, and aortic wall architecture in a validated mouse model of MFS. This protection was independent of hemodynamic effects, as rescue was not achieved in mice treated with beta-adrenergic blockers that showed an equivalent reduction in blood pressure. Members of the Dietz lab went on to show that losartan rescues disease manifestations outside of the cardiovascular system in Marfan mice including developmental emphysema and skeletal muscle myopathy. This work revealed that the congenital myopathy seen in children with severe MFS reflects a TGFbeta-induced failure of muscle regeneration, and that the protection afforded by TGFbeta antagonism extends to mouse models of other conditions including Duchenne muscular dystrophy. As independent investigators, former members of the Dietz lab extended the application of losartan to common human phenotypes including smoking-induced emphysema and disuse muscle atrophy. A number of clinical trials of losartan have been reported and have suggested the potential for protection in children with the most severe presentation of MFS and in subsets of adults with the potential ability to stratify by genotype. In a large prospective trial in children with moderate severity, both losartan and ultra-high dose beta blockers associated with a significant and sustained decline in aortic root z-score over time. While there is clearly more work to be done to define optimal treatment regimens, these data offer the real prospect for medical treatment of MFS and related connective tissue disorders.
The Dietz lab has maintained an aggressive effort to further refine mechanistic understanding of MFS and related disorders. This work demonstrated the importance of so-called noncanonical TGFbeta signaling cascades, prominently including extracellular signal-regulated kinase (ERK) activation. Many lines of evidence implicated ERK as the primary pathogenic driver in the aorta in Marfan mice, including direct and inverse correlation of the level of phosphorylated ERK (pERK) with both disease progression and therapeutic rescue with losartan. Subsequent work showed that treatment of MFS mice with an antagonist of MEK, the ERK kinase, fully normalized aortic root growth rate, aortic wall architecture and survival. Our characterization of the deleterious gene-by-environment interaction imposed by calcium channel blockers (CCBs) in MFS mice again highlighted the importance of ERK and further informed disease mechanism with implication of a TGFbeta-AT1-PLC-IP3-PKC-ERK axis. This led to the hypothesis that treatment with hydralazine (a blocker of IP3-mediated calcium release from the sarcoplasmic reticulum) or PKC antagonists might prove therapeutic; this prediction was realized in Marfan mice in the presence or absence of CCB exposure. Ongoing work by Jef and Alex Doyle and Robert Wardlow has added further granularity to this pathogenic sequence through identification of genetic modifiers of MFS in both mice and people. Jen Habashi has also used this mechanistic foundation to elucidate both the cause and a potential treatment strategy for pregnancy-associated aneurysm tear in MFS and related disorders. Russell Gould and Nicole Wilson are exploring mechanistic ties to a common condition called bicuspid aortic valve with ascending aortic aneurysm.
The conclusion that dysregulated TGFbeta signaling contributes to the multisystem manifestations of MFS and other important disease phenotypes was solidified upon description of a novel human phenotype (later termed Loeys-Dietz syndrome; LDS) that associates some features of MFS (arachnodactyly, scoliosis, chest wall deformity, dural ectasia, aortic root aneurysm) with other unique findings (craniosynostosis, hypertelorism, cleft palate/bifid uvula, cervical spine malformation, club foot deformity, congenital heart disease). Most importantly, people with LDS show aneurysms throughout the arterial tree that tear at younger ages and smaller dimensions when compared to other connective tissue disorders. Work by Bart Loeys showed that LDS is most commonly caused heterozygous missense mutation in the genes encoding either subunit of the TGFbeta receptor (TGFBR1 or TGFBR2). Subsequent work in our lab and by others has shown that LDS can be caused by mutations in other genes encoding TGFbeta effectors including ligands (TGFB2 or TGFB3) or intracellular signaling mediators (SMAD2 or SMAD3). Paradoxically, heterozygous loss-of-function mutations in any of these genes, as seen in LDS, associate with a strong tissue signature for enhanced TGFbeta signaling, and the overt protection afforded by losartan in mouse models of LDS associates with strong attenuation of TGFbeta activity in the aortic wall. Taken together, these apparently contradictory data have engendered considerable controversy in the field regarding the precise role of TGFbeta in the pathogenesis of aortic aneurysm and the therapeutic wisdom of TGFbeta antagonism. Our view that high TGFbeta activity is an important driver of disease was reinforced by the finding of Alex Doyle that Shprintzen-Goldberg syndrome, a condition that includes all features of LDS with the added manifestation of intellectual disability, is caused by loss-of-function mutations in SKI, a prototypical repressor of the TGFbeta transcriptional response. Elena Gallo has arrived at a fully reconciling pathogenic model of aortic root aneurysm through comprehensive consideration of cellular lineages and paracrine interactions in aortic wall morphogenesis and homeostasis.
In another line of investigation, the Dietz lab has taken an interest in scleroderma, defined as progressive hardening of the skin due to excessive collagen deposition. In its most common and severe form called systemic sclerosis (SSc), previously healthy young adults show sudden and unexplained onset of skin and visceral fibrosis in association with the production of autoantibodies. Obstacles to progress in SSc include a poorly defined genetic contribution, the lack of large families to support positional genetic approaches, and the absence of validated animal models. As an alternative, the Dietz lab focused on a rare but Mendelian presentation of scleroderma called stiff skin syndrome (SSS) that shows congenital onset of skin fibrosis with secondary and severe joint contracture. Remarkably, they showed that all families with SSS harbor heterozygous missense mutations in FBN1, the MFS gene, despite the lack of phenotypic overlap between these conditions. Unlike MFS, where mutations occur along the full length of the gene and protein, all mutations causing SSS occur in the sole domain of fibrillin-1 that contains an RGD sequence needed to mediate cell-matrix attachments through integrin binding. The Dietz lab has now made knock-in mouse models of SSS. One line that harbors a naturally occurring SSS mutation shows complete replacement of dermal fat with collagen by 3 months of age. A second line, where the RGD sequence is replaced by RGE, perfectly phenocopies SSS, proving that an isolated and obligate loss of integrin binding to fibrillin-1 is sufficient to initiate and maintain pro-fibrotic programs in the skin. This associates with upregulation of integrin expression in dermal cells and a specific skewing of TGFbeta signaling toward noncanonical (and specifically ERK) cascades. Scleroderma can be prevented in SSS mice through systemic administration of integrin-modulating agents and reversed by treatment with TGFbeta neutralizing antibody. Remarkably, these primary cellular events and therapeutic responses are fully recapitulated in dermal fibroblasts from patients with the more common but less experimentally tractable SSc phenotype. Notably, SSS mice show many of the autoimmune and autoinflammatory findings seen in SSc including autoantibody production and skewing of T-helper cell populations toward pro-inflammatory (Th2 and Th17) subsets. These findings were ultimately linked to the dermal recruitment and activation of plasmacytoid dendritic cells (pDCs) in SSS mice. Either directly or indirectly, pDC activation leads to the production of many pro-fibrotic cytokines including TGFbeta, IL6, IFNalpha and IL17 that can drive the production and/or synthetic repertoire of myofibroblasts. These data have suggested many novel therapeutic targets that are being scrutinized in mouse models.
This work was supported in part by grants from the National Institutes of Health, the National Marfan Foundation, the Smilow Foundation, and the Scleroderma Research Foundation.
As of March 15, 2016