Simultaneous Upregulation of Elastolytic and Elastogenic Factors Are Necessary for Regulated Collateral Diameter Expansion

Background: During arteriogenesis, outward remodeling of the arterial wall expands luminal diameter to produce increased conductance in developing collaterals. We have previously shown that diameter expansion without loss of internal elastic lamina (IEL) integrity requires both degradation of elastic fibers and LOX-mediated repair. The aim of this study was to investigate the expression of genes involved in remodeling of the extracellular matrix (ECM) using a model of arteriogenesis.Methods: Sprague-Dawley rats underwent femoral artery ligation with distal arteriovenous fistula (FAL + AVF) placement. Profunda femoral arteries (PFA) were harvested for analysis at various time points. Serum desmosine, an amino acid found exclusively in elastin, was evaluated with enzyme-linked immunosorbent assay (ELISA) as a marker of tissue elastolysis. Tissue mRNA isolated from FAL + AVF exposed PFAs was compared to the contralateral sham-operated using qPCR. HCAECs were cultured under low shear stress (8 dyn·s/cm2) for 24 h and then exposed to high shear stress (40 dyn·s/cm2) for 2–6 h. Primers used included FBN-1, FBN-2, Timp-2, LOX-1, Trop-E, Cath-K, Cath-S, MMP-2, MMP-9, FBLN-4, and FBLN-5 and were normalized to GAPDH. mRNA fold changes were quantified using the 2-ΔΔCq method. Comparisons between time points were made with non-parametric ANOVA analysis with Bonferroni adjustment.Results: PFAs showed IEL reorganization during arteriogenesis. Serum desmosine levels are significantly elevated at 2 days and one week, with a return to baseline thereafter (p < 0.01). Expression of ECM structural proteins (FBN-1, FBN-2, FBLN-4, FBLN-5, Tropoelastin, TIMP-2, LOX-1) and elastolytic proteins (MMP-2, MMP-9, Cathepsin S, Cathepsin K) exhibited an early peak (p < 0.05) relative to sham PFAs. After two weeks, expression returned to baseline. HCAECs demonstrated upregulation of FBN-2, FBLN-5, LOX-1 and Trop-E at 4 h of high shear stress, as well as elastolytic protein MMP-2.Conclusions: Elastin degradation begins early in arteriogenesis and is mediated by local upregulation of elastolytic genes. Elastolysis appears to be simultaneously balanced by production of elastic fiber components which may facilitate stabilization of the IEL. Endothelial cells are central to initiation of arteriogenesis and begin ECM remodeling in response to altered shear stress.

Experimental aortic aneurysm severity and growth depend on topical elastase concentration and lysyl oxidase inhibition

Abdominal aortic aneurysm (AAA) formation and expansion is highly complex and multifactorial, and the improvement of animal models is an important step to enhance our understanding of AAA pathophysiology. In this study, we explore our ability to influence aneurysm growth in a topical elastase plus β-Aminopropionitrile (BAPN) mouse model by varying elastase concentration and by altering the cross-linking capability of the tissue. To do so, we assess both chronic and acute effects of elastase concentration using volumetric ultrasound. Our results suggest that the applied elastase concentration affects initial elastin degradation, as well as long-term vessel expansion. Additionally, we assessed the effects of BAPN by (1) removing it to restore the cross-linking capability of tissue after aneurysm formation and (2) adding it to animals with stable aneurysms to interrupt cross-linking. These results demonstrate that, even after aneurysm formation, lysyl oxidase inhibition remains necessary for continued expansion. Removing BAPN reduces the aneurysm growth rate to near zero, resulting in a stable aneurysm. In contrast, adding BAPN causes a stable aneurysm to expand. Altogether, these results demonstrate the ability of elastase concentration and BAPN to modulate aneurysm growth rate and severity. The findings open several new areas of investigation in a murine model that mimics many aspects of human AAA.

Sildenafil (Viagra) Aggravates the Development of Experimental Abdominal Aortic Aneurysm

Background cGMP‐hydrolyzing phosphodiesterase type 5 (PDE5) regulates vascular smooth muscle cell (SMC) contraction by antagonizing cGMP‐dependent protein kinase I (PKGI)–dependent SMC relaxation. SMC contractile dysfunction is implicated in the pathogenesis of aortic aneurysm. PDE5 inhibitors have been used for treating erectile dysfunction, such as drug Viagra (sildenafil). However, a few clinical cases have reported the association of Viagra usage with aortic dissection, and reduced PDE5A expression was found in human aortic aneurysm tissues. Therefore, we aimed to investigate the effect of sildenafil on experimental abdominal aortic aneurysm (AAA), the most common form of aortic aneurysm in elderly men. Methods and Results AAA was induced in C57BL/6J male mice by periaortic elastase in combination with blocking elastin/collagen formation via 3‐aminopropionitrile fumarate salt for 35 days. PDE5A protein levels detected by immunostaining were significantly reduced in mouse AAA. Sildenafil application in drinking water significantly aggravated aortic wall dilation and elastin degradation with pre‐existing moderate AAA. The phosphorylation level of myosin light chain 2 at Ser19, a biochemical marker of SMC contraction, was significantly reduced by sildenafil in AAA. Proximity ligation assay further revealed that the interaction between cGMP and PKGI was significantly increased by sildenafil in AAA, suggesting an elevation of PKGI activation in AAA. Conclusions Sildenafil treatment aggravated the degradation of elastin fibers and progression of experimental AAA by dysregulating cGMP and contractile signaling in SMCs. Our findings may raise the caution of clinical usage of Viagra in aneurysmal patients.

Syndecan-1 is overexpressed in human thoracic aneurysm but is dispensable for the disease progression in vivo

Glycosaminoglycans (GAGs) pooling has been considered since long as one of the histopathological characteristics defining thoracic aortic aneurysm (TAA) together with smooth muscle cells (SMCs) apoptosis and elastin fibers degradation. However, few information is provided about GAGs composition or potential implication in TAA pathology. Syndecan-1 (Sdc-1) is a heparan sulfate proteoglycan that is implicated in extracellular matrix (ECM) interaction and assembly, regulation of SMCs phenotype and various aspects of inflammation in the vascular wall. In the current work, the regulation of Sdc-1 protein was examined in human TAA by ELISA and immunohistochemistry. In addition, the role of Sdc-1 was evaluated in descending TAA in vivo using a mouse model combining both aortic wall weakening and hypertension. Our results showed that Sdc-1 protein is over expressed in human TAA aortas compared to healthy counterparts and that SMCs are the major cell type expressing Sdc-1. Similarly, in the mouse model used, Sdc-1 expression was increased in TAA aortas compared to healthy samples. Although its protective role against abdominal aneurysm has been reported, we observed that Sdc-1 was dispensable for TAA prevalence or rupture. In addition, Sdc-1 deficiency did not alter the extent of aortic wall dilatation, elastin degradation, collagen deposition, or leukocyte recruitment in our TAA model. These findings suggest that Sdc-1 could be a biomarker revealing TAA pathology. Future investigations could uncover the underlying mechanisms leading to Sdc-1 expression alteration in TAA.

Elastin degradation markers are elevated in never-smokers with past history of prolonged exposure to secondhand tobacco smoke and are inversely associated with their lung function

Background- Prolonged past exposure to secondhand tobacco smoke (SHS) in never-smokers is associated with occult obstructive lung disease and abnormal lung function, in particular reduced diffusing capacity. Previous studies have shown ongoing SHS exposure to be associated with increased elastin degradation markers (EDM) desmosine and isodesmosine. Research Question- Are EDM levels elevated in persons with remote history of SHS exposure, and are those levels associated with reduced lung function? Study Design and Methods- We measured the plasma levels of EDM from 193 never-smoking flight attendants with history of remote but prolonged SHS exposure in aircraft cabin and 103 nonsmoking flight attendants or sea-level control participants without history of cabin SHS exposure, and examined those levels versus their lung function with adjustment for covariates. The cabin SHS exposure was estimated based on airline employment history and dates of smoking ban enactment. EDM plasma levels were quantified by high-performance liquid chromatography and tandem mass spectrometry. Results- The median [interquartile range; IQR] plasma EDM level for all participants was 0.30 [0.24 to 0.36] ng/mL with a total range of 0.16 to 0.65 ng/mL. Plasma EDM levels were elevated in those with history of exposure to cabin SHS compared to those not exposed (0.33±0.08 vs. 0.26±0.06 ng/mL; age- and sex-adjusted P<0.001). In those with history of cabin SHS-exposure, higher EDM levels were associated with lower diffusing capacity (parameter estimate (PE) [95%CI]=4.2 [0.4 to 8.0] %predicted decrease per 0.1 ng/mL increase in EDM; P=0.030). Furthermore, EDM levels were inversely associated with FEV1, FEV1/FVC, and FEF25-75 (PE [95%CI]=5.8 [2.1 to 9.4], 4.0 [2.2 to 5.7], and 12.5 [5.8 to 19.2]% predicted decrease per 0.1 ng/mL increase in EDM, respectively) (P<0.001).

Temporal and Quantitative Analysis of Aortic Immunopathologies in Elastase-Induced Mouse Abdominal Aortic Aneurysms

Objective. Elastase-induced abdominal aortic aneurysm (AAA) model is widely used for aneurysmal pathogenesis and translational research. However, temporal alternations in aneurysmal histologies remain unknown. This study is aimed at analyzing temporal immunopathologies of aneurysmal aorta following experimental AAA induction. Methods. Male C57BL/6J mice at the age of 10-14 weeks received intra-aortic infusion of elastase to induce AAAs. Aortic diameters at the baseline and indicated days after AAA induction were measured, and aortae were collected for histopathological analysis. Results. Aorta diameters increased from 0.52 mm at the baseline levels to 0.99 mm, 1.34 mm, and 1.41 mm at days 7, 14, and 28, respectively, corresponding 90%, 158%, and 171% increases over the baseline level. Average aortic diameters did not differ between days 14 and 28. Severe elastin degradation and smooth muscle cell depletion were found at days 14 and 28 as compared to the baseline and day 7. No difference in the scores of medial elastin and SMC destruction was noted between days 14 and 28. Consistent results were found for leukocyte accumulation, neoangiogenesis, and matrix metalloproteinase expression. Twenty-eight days after AAA induction, all aneurysmal pathologies showed an attenuated trend, although most histopathological parameters did no differ between days 14 and 28. Conclusion. Our data suggest that almost aneurysmal immunohistopathologies reach maximal 14 days following AAA induction. Analysis of day 14 histologies is sufficient for AAA pathogenesis and translational studies in elastase-induced mouse experimental AAAs.

MMP-2 gene silencing attenuates age-dependent carotid stiffness via reduction of elastin degradation and increased eNOS activation

Background and aims Arterial stiffness is a hallmark of vascular aging. Being characterized by a loss of elasticity of large arterial walls, arterial stiffness is associated with an increased risk of cardiovascular disease (CVD). The age-dependent arterial stiffness is primarily attributed to alterations in the elastic and collagen deposition that is regulated by a number of enzymes, including matrix metalloproteinase-2 (MMP-2). Nevertheless, the mechanistic link between age-dependent arterial stiffness and MMP-2 remains unclear. In this study, we investigated the effect and efficacy of therapeutic MMP-2 knockdown using small interfering RNA (siRNA) on age-dependent arterial stiffness. Methods Pulse wave velocity (PWV) was assessed in the right carotid artery of wild-type (WT) mice of different age groups. MMP-2 levels and activity in the carotid artery and plasma of young (3 months) and aged (20–25 months) WT mice were determined. Old WT mice (18–21 months) were treated for 4 weeks with either MMP-2 or scrambled siRNA, in which carotid PWV was assessed at baseline, 2 and 4 weeks after the start of the treatment. Elastin to collagen ratio, desmosin (DES) level, and endothelial nitric oxide synthase (eNOS) pathways were also evaluated and compared. Lastly, levels of circulating MMP-2 and DES, the breakdown product of elastin, were measured in a human cohort (23–86 years old), in whom carotid-femoral PWV was assessed. Results Carotid PWV, as well as both vascular and circulating MMP-2 levels, were elevated with increasing age in WT mice (Figure 1). Therapeutic MMP-2 knockdown in aged WT mice reduced the vascular MMP-2 expression and attenuated age-dependent carotid stiffness. Increased elastin to collagen ratio and a lower plasma DES level were observed on MMP-2 silenced treated animals (Figure 2). Moreover, siMMP-2 treated mice showed enhanced eNOS phosphorylation on Ser1177. A direct interaction between MMP-2 and eNOS was also observed, which, interestingly, is augmented with age. Finally, collected human data showed a higher level of circulating MMP-2 levels on the elderly subjects. In addition, plasma DES level is positively correlated with age and aortic PWV, indicating the involvement of vascular elastin catabolism on arterial stiffness. Conclusions Therapeutic MMP-2 gene silencing, specifically targeting vascular MMP-2, attenuates age-dependent carotid stiffness. This effect is mediated by augmenting eNOS activation and reducing elastin degradation. Thus, our findings indicate MMP-2 as a potential therapeutic target to mitigate age-dependent arterial stiffness and CVD. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swiss National Science Foundation,Foundation for Cardiovascular Research–Zurich Heart House Figure 1

No Effect of Hypercholesterolemia on Elastase-Induced Experimental Abdominal Aortic Aneurysm Progression

Objective: Epidemiological studies link hyperlipidemia with increased risk for abdominal aortic aneurysms (AAAs). However, the influence of lipid-lowering drugs statins on prevalence and progression of clinical and experimental AAAs varies between reports, engendering controversy on the association of hyperlipidemia with AAA disease. This study investigated the impact of hypercholesterolemia on elastase-induced experimental AAAs in mice. Methods: Both spontaneous (targeted deletion of apolipoprotein E) and induced mouse hypercholesterolemia models were employed. In male wild type (WT) C57BL/6J mice, hypercholesterolemia was induced via intraperitoneal injection of an adeno-associated virus (AAV) encoding a gain-of-function proprotein convertase subtilisin/kexin type 9 mutation (PCSK9) followed by the administration of a high-fat diet (HFD) (PCSK9+HFD) for two weeks. As normocholesterolemic controls for PCSK9+HFD mice, WT mice were infected with PCSK9 AAV and fed normal chow, or injected with phosphate-buffered saline alone and fed HFD chow. AAAs were induced in all mice by intra-aortic infusion of porcine pancreatic elastase and assessed by ultrasonography and histopathology. Results: In spontaneous hyper- and normo-cholesterolemic male mice, the aortic diameter enlarged at a constant rate from day 3 through day 14 following elastase infusion. AAAs, defined as a more than 50% diameter increase over baseline measurements, formed in all mice. AAA progression was more pronounced in male mice, with or without spontaneous hyperlipidemia. The extent of elastin degradation and smooth muscle cell depletion were similar in spontaneous hyper- (score 3.5 for elastin and 4.0 for smooth muscle) and normo- (both scores 4.0) cholesterolemic male mice. Aortic mural macrophage accumulation was also equivalent between the two groups. No differences were observed in aortic accumulation of CD4+ or CD8+ T cells, B cells, or mural angiogenesis between male spontaneous hyper- and normocholesterolemic mice. Similarly, no influence of spontaneous hypercholesterolemia on characteristic aneurysmal histopathology was noted in female mice. In confirmatory experiments, induced hypercholesterolemia also exerted no appreciable effect on AAA progression and histopathologies. Conclusion: This study demonstrated no recognizable impact of hypercholesterolemia on elastase-induced experimental AAA progression in both spontaneous and induced hypercholesterolemia mouse models. These results add further uncertainty to the controversy surrounding the efficacy of statin therapy in clinical AAA disease.

Aortic Stiffness Hysteresis in Isolated Mouse Aortic Segments Is Intensified by Contractile Stimuli, Attenuated by Age, and Reversed by Elastin Degradation

Aim: Cyclic stretch of vascular tissue at any given pressure reveals greater dimensions during unloading than during loading, which determines the cardiac beat-by-beat hysteresis loop on the pressure-diameter/volume relationship. The present study did not focus on hysteresis during a single stretch cycle but investigated whether aortic stiffness determined during continuous stretch at different pressures also displayed hysteresis phenomena.Methods: Aortic segments from C57Bl6 mice were mounted in the Rodent Oscillatory Set-up for Arterial Compliance (ROTSAC), where they were subjected to high frequency (10 Hz) cyclic stretch at alternating loads equivalent to a constant theoretical pulse pressure of 40 mm Hg. Diastolic and systolic diameter, compliance, and the Peterson elastic modulus (Ep), as a measure of aortic stiffness, was determined starting at cyclic stretch between alternating loads corresponding to 40 and 80 mm Hg, at each gradual load increase equivalent to 20 mm Hg, up to loads equivalent to pressures of 220 and 260 mm Hg (loading direction) and then repeated in the downward direction (unloading direction). This was performed in baseline conditions and following contraction by α1 adrenergic stimulation with phenylephrine or by depolarization with high extracellular K+ in aortas of young (5 months), aged (26 months) mice, and in segments treated with elastase.Results: In baseline conditions, diastolic/systolic diameters and compliance for a pulse pressure of 40 mm Hg were larger at any given pressure upon unloading (decreasing pressure) than loading (increasing pressure) of the aortic segments. The pressure-aortic stiffness (Ep) relationship was similar in the loading and unloading directions, and aortic hysteresis was absent. On the other hand, hysteresis was evident after activation of the VSMCs with the α1 adrenergic agonist phenylephrine and with depolarization by high extracellular K+, especially after inhibition of basal NO release with L-NAME. Aortic stiffness was significantly smaller in the unloading than in the loading direction. In comparison with young mice, old-mouse aortic segments also displayed contraction-dependent aortic hysteresis, but hysteresis was shifted to a lower pressure range. Elastase-treated segments showed higher stiffness upon unloading over nearly the whole pressure range.Conclusions: Mouse aortic segments display pressure- and contraction-dependent diameter, compliance, and stiffness hysteresis phenomena, which are modulated by age and VSMC-extracellular matrix interactions. This may have implications for aortic biomechanics in pathophysiological conditions and aging.

Targeted delivery of pentagalloyl glucose inhibits matrix metalloproteinase activity and preserves elastin in emphysematous lungs

Background Elastin degradation has been established as one of the driving factors of emphysema. Elastin-derived peptides (EDPs) are shown to act as a chemoattractant for monocytes. Effectively shielding elastin from elastolytic damage and regenerating lost elastin are two important steps in improving the mechanical function of damaged lungs. Pentagalloyl glucose (PGG) has been shown to preserve elastin in vascular tissues from elastolytic damage in vivo and aid in elastin deposition in vitro. Methods We created emphysema by elastase inhalation challenge in mice. Albumin nanoparticles loaded with PGG, conjugated with elastin antibody, were delivered to target degraded elastin in lungs. We investigated matrix metalloproteinase-12 activity and lung damage by measuring dynamic compliance and tidal volume changes. Results Ex-vivo experiments demonstrated elastin preservation in PGG treated samples compared to controls. Inhaled nanoparticles conjugated with elastin antibody retained for extended periods in lungs. Further, mice treated with PGG nanoparticles showed a significant suppression of MMP-12 activity measured in the lungs. We observed suppression of emphysema in terms of dynamic lung compliance and tidal volume change compared to the control group. The histological examination further confirmed elastin preservation in the lungs. Conclusion These results demonstrate successful targeted delivery of nanoparticles loaded with PGG to inhibit MMP-12 activity and preserve elastin in the lungs. Such targeted PGG therapy has potential therapeutic use in the management of emphysema.