Jeffrey A. Jones

Transforming growth factor-beta signaling in thoracic aortic aneurysm development: a paradox in pathogenesis

Thoracic aortic aneurysms (TAAs) are potentially devastating, and due to their asymptomatic behavior, pose a serious health risk characterized by the lack of medical treatment options and high rates of surgical morbidity and mortality. Independent of the inciting stimuli (biochemical/mechanical), TAA development proceeds by a multifactorial process influenced by both cellular and extracellular mechanisms, resulting in alterations of the structure and composition of the vascular extracellular matrix (ECM). While the role of enhanced ECM proteolysis in TAA formation remains undisputed, little attention has been focused on the upstream signaling events that drive the remodeling process. Recent evidence highlighting the dysregulation of transforming growth factor-β (TGF-β) signaling in ascending TAAs from Marfan syndrome patients has stimulated an interest in this intracellular signaling pathway. However, paradoxical discoveries have implicated both enhanced TGF-β signaling and loss of function TGF-β receptor mutations, in aneurysm formation; obfuscating a clear functional role for TGF-β in aneurysm development. In an effort to elucidate this subject, TGF-β signaling and its role in vascular remodeling and pathology will be reviewed, with the aim of identifying potential mechanisms of how TGF-β signaling may contribute to the formation and progression of TAA.

Positively Charged Ceramide Is a Potent Inducer of Mitochondrial Permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H+ channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.

Regional Heterogeneity within the Aorta: Relevance to Aneurysm Disease

Vascular remodeling within the aorta results in a loss of structural integrity with consequent aneurysm formation. This degradation is more common in the abdominal aorta but also occurs above the diaphragm in the thoracic aorta. Conventionally, the aorta has been considered a large vascular conduit with uniform cellular and extracellular structure and function. Evidence is accumulating, however, to suggest that variations exist between the thoracic and abdominal aorta, thereby demonstrating regional heterogeneity. Further pathophysiologic studies of aortic dilation in each of these regions have identified disparities in atherosclerotic plaque deposition, vessel mechanics, protease profiles, and cell-signaling pathways. Improved understanding of this spatial heterogeneity might promote evolution in the management of aneurysm disease through computational models of aortic wall stress, imaging of proteolytic activity, targeted pharmacologic treatment, and application of region-specific gene therapy.

Selective MicroRNA Suppression in Human Thoracic Aneurysms Relationship of miR-29a to Aortic Size and Proteolytic Induction

Background— Increasing evidence points to a direct role for altered microRNA (miRNA or miR) expression levels in cardiovascular remodeling and disease progression. Although alterations in miR expression levels have been directly linked to cardiac hypertrophy, fibrosis, and remodeling, their role in regulating gene expression during thoracic aortic aneurysm (TAA) development has yet to be explored. Methods and Results— The present study examined miR expression levels in aortic tissue specimens collected from patients with ascending TAAs by quantitative real-time PCR, and observed decreased miR expression (miRs -1, -21, -29a, -133a, and -486) as compared with normal aortic specimens. A significant relationship between miR expression levels (miRs -1, -21, -29a, and -133a) and aortic diameter was identified; as aortic diameter increased, miR expression decreased. Through the use of a bioinformatics approach, members of the matrix metalloproteinase (MMP) family, proteins involved in TAA development, were examined for putative miR binding sites. MMP-2 and MMP-9 were identified as potential targets for miR-29a and miR-133a, respectively, and MMP-2 was subsequently verified as a miR-29a target in vitro. A significant inverse relationship between miR-29a and total MMP-2 was then identified in the clinical TAA specimens. Conclusions— These findings demonstrate altered miR expression patterns in clinical TAA specimens, suggesting that the loss of specific miR expression may allow for the elaboration of specific MMPs capable of driving aortic remodeling during TAA development. Importantly, these data suggest that these miRs have biological and clinical relevance to the behavior of TAAs and may provide significant targets for therapeutic and diagnostic applications.Background— Increasing evidence points to a direct role for altered microRNA (miRNA or miR) expression levels in cardiovascular remodeling and disease progression. Although alterations in miR expression levels have been directly linked to cardiac hypertrophy, fibrosis, and remodeling, their role in regulating gene expression during thoracic aortic aneurysm (TAA) development has yet to be explored. Methods and Results— The present study examined miR expression levels in aortic tissue specimens collected from patients with ascending TAAs by quantitative real-time PCR, and observed decreased miR expression (miRs -1, -21, -29a, -133a, and -486) as compared with normal aortic specimens. A significant relationship between miR expression levels (miRs -1, -21, -29a, and -133a) and aortic diameter was identified; as aortic diameter increased, miR expression decreased. Through the use of a bioinformatics approach, members of the matrix metalloproteinase (MMP) family, proteins involved in TAA development, were examined for putative miR binding sites. MMP-2 and MMP-9 were identified as potential targets for miR-29a and miR-133a, respectively, and MMP-2 was subsequently verified as a miR-29a target in vitro. A significant inverse relationship between miR-29a and total MMP-2 was then identified in the clinical TAA specimens. Conclusions— These findings demonstrate altered miR expression patterns in clinical TAA specimens, suggesting that the loss of specific miR expression may allow for the elaboration of specific MMPs capable of driving aortic remodeling during TAA development. Importantly, these data suggest that these miRs have biological and clinical relevance to the behavior of TAAs and may provide significant targets for therapeutic and diagnostic applications.

Aortic dilatation with bicuspid aortic valves: cusp fusion correlates to matrix metalloproteinases and inhibitors.

BACKGROUND Congenital bicuspid aortic valves (BAVs) result from fusion of 2 valve cusps, resulting in left-noncoronary (L-N), right-left (R-L), and right-noncoronary (R-N) morphologic presentations. BAVs predispose to ascending thoracic aortic aneurysms (ATAAs). This study hypothesized that ATAAs with each BAV morphologic group possess unique signatures of matrix metalloproteinases (MMPs) and endogenous tissue inhibitors of metalloproteinases (TIMPs). METHODS Ascending thoracic aortic aneurysm tissue from 46 patients with BAVs was examined for MMP/TIMP abundance, and global MMP activity was compared with normal aortic specimens (n=15). Proteolytic balance was calculated as the ratio of MMP abundance to a composite TIMP score. Results were stratified by valve morphologic group (L-N [n=6], R-L [n=31], and R-N [n=9]). RESULTS The BAV specimens (p<0.05 versus normal aorta, 100%) displayed elevated global MMP activity (273%±63%), MMP-9 (263%±47%), and decreased MMP-7 (56%±10%), MMP-8 (58%±11%), TIMP-1 (63%±7%), and TIMP-4 (38%±3%). The R-L group showed increased global MMP activity (286%±89%) and MMP-9 (267%±55%) with reduced MMP-7 (45%±7%), MMP-8 (68%±15%), TIMP-1 (58%±7%), and TIMP-4 (35%±3%). The L-N group showed elevated global MMP activity (284%±71%) and decreased MMP-8 (37%±17%) and TIMP-4 (48%±14) activity. In the R-N group, MMP-7 (46%±13%) and MMP-8 (36%±17%) and TIMP-1 (59%±10%) and TIMP-4 (42%±5%) were decreased. The R-L group demonstrated an increased proteolytic balance for MMP-1, MMP-9, and MMP-12 relative to L-N and R-N. CONCLUSIONS Each BAV morphologic group possesses a unique signature of MMPs and TIMPs. MMP/TIMP score ratios suggest that the R-L group may be more aggressive, justifying earlier surgical intervention.

Alterations in Aortic Cellular Constituents during Thoracic Aortic Aneurysm Development: Myofibroblast-Mediated Vascular Remodeling

The present study tested the hypothesis that changes in the resident endogenous cellular population accompany alterations in aortic collagen and elastin content during thoracic aortic aneurysm (TAA) development in a murine model. Descending thoracic aortas were analyzed at various time points (2, 4, 8, and 16 weeks) post-TAA induction (0.5 M CaCl2, 15 minutes). Aortic tissue sections were subjected to histological staining and morphometric analysis for collagen and elastin, as well as immunostaining for cell-type-specific markers to quantify fibroblasts, myofibroblasts, and smooth-muscle cells. Results were compared with reference control mice processed in the same fashion. Aortic dilatation was accompanied by changes in the elastic architecture that included: a decreased number of elastic lamellae (from 6 to 4); altered area fraction of elastin (elevated at 4 weeks and decreased at 16 weeks); and a decreased area between elastic lamellae (minimum reached at 4 weeks). Total collagen content did not change over time. Increased immunoreactivity for fibroblast and myofibroblast markers was observed at 8- and 16-week post-TAA-induction, whereas immunoreactivity for smooth-muscle cell markers peaked at 4 weeks and returned to baseline by 16 weeks. Therefore, this study demonstrated that changes in aortic elastin content were accompanied by the emergence of a subset of fibroblast-derived myofibroblasts whose altered phenotype may play a significant role in TAA development through the enhancement of extracellular matrix proteolysis.

Differential Effects of Mechanical and Biological Stimuli on Matrix Metalloproteinase Promoter Activation in the Thoracic Aorta

Background— The effect of multiple integrated stimuli on vascular wall expression of matrix metalloproteinases (MMPs) remains unknown. Accordingly, this study examined the influence of the vasoactive peptide angiotensin II (Ang II) on wall tension–induced promoter activation of MMP-2, MMP-9, and membrane type-1 MMP (MT1-MMP). Methods and Results— Thoracic aortic rings harvested from transgenic reporter mice containing the MMP-2, MMP-9, or MT1-MMP promoter sequence fused to a reporter gene were subjected to 3 hours of wall tension at 70, 85, or 100 mm Hg, with or without 100 nM Ang II. Total RNA was harvested from the aortic rings, and reporter gene transcripts were quantified by quantitative real-time polymerase chain reaction to measure MMP promoter activity. MT1-MMP promoter activity was increased at both 85 and 100 mm Hg, compared with baseline tension of 70 mm Hg, whereas treatment with Ang II stimulated MT1-MMP promoter activity to the same degree at all tension levels (P<0.05). Elevated tension and Ang II displayed a potential synergistic enhancement of MMP-2 promoter activation at 85 and 100 mm Hg, whereas the same stimuli caused a decrease in MMP-9 promoter activity (P<0.05) at 100 mm Hg. Conclusions— This study demonstrated that exposure to a relevant biological stimulus (Ang II) in the presence of elevated tension modulated MMP promoter activation. Furthermore, these data suggest that a mechanical-molecular set point exists for the induction of MMP promoter activation and that this set point can be adjusted up or down by a secondary biological stimulus. Together, these results may have significant clinical implications toward the regulation of hypertensive vascular remodeling.

Pressure overload-dependent membrane type 1-matrix metalloproteinase induction: relationship to LV remodeling and fibrosis

Increased myocardial extracellular matrix collagen represents an important structural milestone during the development of left ventricular (LV) pressure overload (PO); however, the proteolytic pathways that contribute to this process are not fully understood. This study tested the hypothesis that membrane type 1-matrix metalloproteinase (MT1-MMP) is directly induced at the transcriptional level in vivo during PO and is related to changes in LV collagen content. PO was induced in vivo by transverse aortic constriction in transgenic mice containing the full length human MT1-MMP promoter region ligated to luciferase (MT1-MMP Prom mice). MT1-MMP promoter activation (luciferase expression), expression, and activity; collagen volume fraction (CVF); and left atrial dimension were measured at 1 (n = 8), 2 (n = 12), and 4 (n = 17) wk following PO. Non-PO mice (n = 10) served as controls. Luciferase expression increased by fivefold at 1 wk, fell at 2 wk, and increased again by ninefold at 4 wk of PO (P < 0.05). MT1-MMP expression and activity increased at 1 wk, fell at 2 wk, and increased again at 4 wk after PO. CVF increased at 1 wk, remained unchanged at 2 wk, and increased by threefold at 4 wk of PO (P < 0.05). There was a strong positive correlation between CVF and MT1-MMP activity (r = 0.80, P < 0.05). Left atrial dimension remained unchanged at 1 and 2 wk but increased by 25% at 4 wk of PO. When a mechanical load was applied in vitro to LV papillary muscles isolated from MT1-MMP Prom mice, increased load caused MT1-MMP promoter activation to increase by twofold and MT1-MMP expression to increase by fivefold (P < 0.05). These findings challenge the canonical belief that PO suppresses overall matrix proteolytic activity, but rather supports the concept that certain proteases, such as MT1-MMP, play a pivotal role in PO-induced matrix remodeling and fibrosis.

Regular ArticlesAlterations in Aortic Cellular Constituents during Thoracic Aortic Aneurysm Development: Myofibroblast-Mediated Vascular Remodeling

The present study tested the hypothesis that changes in the resident endogenous cellular population accompany alterations in aortic collagen and elastin content during thoracic aortic aneurysm (TAA) development in a murine model. Descending thoracic aortas were analyzed at various time points (2, 4, 8, and 16 weeks) post-TAA induction (0.5 M CaCl2, 15 minutes). Aortic tissue sections were subjected to histological staining and morphometric analysis for collagen and elastin, as well as immunostaining for cell-type-specific markers to quantify fibroblasts, myofibroblasts, and smooth-muscle cells. Results were compared with reference control mice processed in the same fashion. Aortic dilatation was accompanied by changes in the elastic architecture that included: a decreased number of elastic lamellae (from 6 to 4); altered area fraction of elastin (elevated at 4 weeks and decreased at 16 weeks); and a decreased area between elastic lamellae (minimum reached at 4 weeks). Total collagen content did not change over time. Increased immunoreactivity for fibroblast and myofibroblast markers was observed at 8- and 16-week post-TAA-induction, whereas immunoreactivity for smooth-muscle cell markers peaked at 4 weeks and returned to baseline by 16 weeks. Therefore, this study demonstrated that changes in aortic elastin content were accompanied by the emergence of a subset of fibroblast-derived myofibroblasts whose altered phenotype may play a significant role in TAA development through the enhancement of extracellular matrix proteolysis.

Altered Transforming Growth Factor-Beta Signaling in a Murine Model of Thoracic Aortic Aneurysm

Objective: Thoracic aortic aneurysms (TAAs) develop by a multifactorial process involving maladaptive signaling pathways that alter the aortic vascular environment. Transforming growth factor-beta (TGF-β) has been implicated in regulating the structure and composition of the extracellular matrix by differential activation of various intracellular signaling pathways. However, whether and to what degree TGF-β signaling contributes to TAA development remains unclear. Accordingly, the hypothesis that alterations in TGF-β signaling occur during aneurysm formation was tested in a murine model of TAA. Methods: TAAs were surgically induced in mice (C57BL/6J) and aortas were analyzed at predetermined time points (1, 2, and 4 weeks post-TAA induction). Quantitative real-time PCR (QPCR) was performed to evaluate the expression of 84 relevant TGF-β superfamily genes, and the protein levels of key signaling intermediates were measured by immunoblotting. Results were compared to unoperated reference control mice. Results: QPCR revealed increased expression of TGF-β superfamily ligands (Gdf-2, -6, -7, Inhba), ligand inhibitors (Bmper, Chrd, Gsc), and transcriptional regulators (Dlx2, Evi1), among other genes (Cdkn2b, Igf1, IL-6). Protein levels of TGF-β receptorII, Smad2, Smad1/5/8, phospho-Smad1/5/8, and Smurf1 were increased from control values post-TAA induction. Both TGF-β receptorI and Smad4 were decreased from control values, while ALK-1 levels remained unchanged. Conclusions: These alterations in the TGF-β pathway suggest a mechanism by which primary signaling is switched from a TGF-βRI/Smad2-dependent response, to an ALK-1/Smad1/5/8 response, representing a significant change in signaling outcome, which may enhance matrix degradation.