Inhibition of the Renin-Angiotensin System Fails to Suppress ß-Aminopropionitrile-Induced Thoracic Aortopathy in Mice-Brief Report.

BACKGROUND: Cross-linking of lysine residues in elastic and collagen fibers is a vital process in aortic development. Inhibition of lysyl oxidase by BAPN (ß-aminopropionitrile) leads to thoracic aortopathies in mice. Although the renin-angiotensin system contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the renin-angiotensin system protects against aortopathy caused by the impairment of elastic fiber/collagen crosslinking. METHODS: BAPN (0.5% wt/vol) was started in drinking water to induce aortopathies in male C57BL/6J mice at 4 weeks of age for 4 weeks. Five approaches were used to investigate the impact of the renin-angiotensin system. Bulk RNA sequencing was performed to explore potential molecular mechanisms of BAPN-induced thoracic aortopathies. RESULTS: Losartan increased plasma renin concentrations significantly, compared with vehicle-infused mice, indicating effective angiotensin II type 1 receptor inhibition. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor of the renin-angiotensin system, irbesartan, an insurmountable inhibitor, was also tested. Although increased plasma renin concentrations indicated effective inhibition, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin II type 1 receptor blockade. Next, we inhibited angiotensin II production by pharmacological or genetic depletion of AGT (angiotensinogen), the unique precursor of angiotensin II. However, neither suppressed BAPN-induced thoracic aortic rupture and dilatation. Aortic RNA sequencing revealed molecular changes during BAPN administration that were distinct from other types of aortopathies in which angiotensin II type 1 receptor inhibition protects against aneurysm formation. CONCLUSIONS: Inhibition of either angiotensin II action or production of the renin-angiotensin system does not attenuate BAPN-induced thoracic aortopathies in mice.

Microenergy acoustic pulse therapy restores urethral wall integrity and continence in a rat model of female stress incontinence.

OBJECTIVE: To determine the outcomes and mechanisms of microenergy acoustic pulse (MAP) therapy in an irreversible rat model of female stress urinary incontinence. MATERIALS AND METHODS: Twenty-four female Sprague-Dawley rats were randomly assigned into four groups: sham control (sham), vaginal balloon dilation and ovariectomy (VBDO), VBDO + ß-aminopropionitrile (BAPN), and VBDO + ß-aminopropionitrile treated with MAP (MAP). MAP therapy was administered twice per week for 4 weeks. After a 1-week washout period, all 24 rats were evaluated with functional and histological studies. The urethral vascular plexus was examined by immunofluorescence staining with antibodies against collagen IV and von Willebrand factor (vWF). The urethral smooth muscle stem/progenitor cells (uSMPCs) were isolated and functionally studied in vivo and in vitro. RESULTS: Functional study with leak point pressure (LPP) measurement showed that the MAP group had significantly higher LPPs compared to VBDO and BAPN groups. MAP ameliorated the decline in urethral wall thickness and increased the amount of extracellular matrix within the urethral wall, especially in the urethral and vaginal elastic fibers. MAP also improved the disruption of the urethral vascular plexus in the treated animals. In addition, MAP enhanced the regeneration of urethral and vaginal smooth muscle, and uSMPCs could be induced by MAP to differentiate into smooth muscle and neuron-like cells in vitro. CONCLUSION: MAP appears to restore urethral wall integrity by increasing muscle content in the urethra and the vagina and by improving the urethral vascular plexus and the extracellular matrix.

Moderate aerobic exercise prevents matrix degradation and death in a mouse model of aortic dissection and aneurysm.

Thoracic aortic aneurysm and dissection (TAAD) is a deadly disease characterized by intimal disruption induced by hemodynamic forces of the circulation. The effect of exercise in patients with TAAD is largely unknown. ß-Aminopropionitrile (BAPN) is an irreversible inhibitor of lysyl oxidase that induces TAAD in mice. The objective of this study was to investigate the effect of aerobic exercise on BAPN-induced TAAD. Upon weaning, mice were given either BAPN-containing water or standard drinking water and subjected to either conventional cage activity (BAPN-CONV) or forced treadmill exercise (BAPN-EX) for up to 26 wk. Mortality was 23.5% (20/85) for BAPN-CONV mice versus 0% (0/22) for BAPN-EX mice (hazard ratio 3.8; P = 0.01). BAPN induced significant elastic lamina fragmentation and intimal-medial thickening compared with BAPN-untreated controls, and aneurysms were identified in 50% (5/10) of mice that underwent contrast-enhanced CT scanning. Exercise significantly decreased BAPN-induced wall thickening, calculated circumferential wall tension, and lumen diameter, with 0% (0/5) of BAPN-EX demonstrating chronic aortic aneurysm formation on CT scan. Expression of selected genes relevant to vascular diseases was analyzed by qRT-PCR. Notably, exercise normalized BAPN-induced increases in TGF-ß pathway-related genes Cd109, Smad4, and Tgfßr1; inflammation-related genes Vcam1, Bcl2a1, Ccr2, Pparg, Il1r1, Il1r1, Itgb2, and Itgax; and vascular injury- and response-related genes Mmp3, Fn1, and Vwf. Additionally, exercise significantly increased elastin expression in BAPN-treated animals compared with controls. This study suggests that moderate aerobic exercise may be safe and effective in preventing the most devastating outcomes in TAAD.NEW & NOTEWORTHY Moderate aerobic exercise was shown to significantly reduce mortality, extracellular matrix degradation, and thoracic aortic aneurysm and dissection formation associated with lysyl oxidase inhibition in a mouse model. Gene expression suggested a reversal of TGF-ß, inflammation, and extracellular matrix remodeling pathway dysregulation, along with augmented elastogenesis with exercise.

Inhibition of lysyl oxidase stimulates TGF-ß signaling and metalloproteinases-2 and -9 expression and contributes to the disruption of ascending aorta in rats: protection by propylthiouracil.

Mutations in lysyl oxidase (LOX) genes cause severe vascular anomalies in mice and humans. LOX activity can be irreversibly inhibited by the administration of ß-aminoproprionitrile (BAPN). We investigated the mechanisms underlying the damage to the ascending thoracic aorta induced by LOX deficiency and evaluated whether 6-propylthiouracil (PTU) can afford protection in rats. BAPN administration caused disruption of the ascending aortic wall, increased the number of apoptotic cells, stimulated TGF-ß signaling (increase of nuclear p-SMAD2 staining), and up-regulated the expression of metalloproteinases-2 and -9. In BAPN-treated animals, PTU reduced apoptosis, p-SMAD2 staining, MMP-2, and -9 expression, and markedly decreased the damage to the aortic wall. Our results suggest that, as in some heritable vascular diseases, enhanced TGF-ß signaling and upregulation of MMP-2 and -9 can contribute to the pathogenesis of ascending aorta damage caused by LOX deficiency. We have also shown that PTU, a drug already in clinical use, protects against the effects of LOX inhibition. MMP-2 and -9 might be potential targets of new therapeutic strategies for the treatment of vascular diseases caused by LOX deficiency.

Increased Collagen Crosslinking in Stiff Clubfoot Tissue: Implications for the Improvement of Therapeutic Strategies.

Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)-mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast-like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, ß-aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell-populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10-40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell-mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.

Substrate Strain Mitigates Effects of ß-Aminopropionitrile-Induced Reduction in Enzymatic Crosslinking.

Enzymatic crosslinks stabilize type I collagen and are catalyzed by lysyl oxidase (LOX), a step interrupted through ß-aminopropionitrile (BAPN) exposure. This study evaluated dose-dependent effects of BAPN on osteoblast gene expression of type I collagen, LOX, and genes associated with crosslink formation. The second objective was to characterize collagen produced in vitro after exposure to BAPN, and to explore changes to collagen properties under continuous cyclical substrate strain. To evaluate dose-dependent effects, osteoblasts were exposed to a range of BAPN dosages (0-10 mM) for gene expression analysis and cell proliferation. Results showed significant upregulation of BMP-1, POST, and COL1A1 and change in cell proliferation. Results also showed that while the gene encoding LOX was unaffected by BAPN treatment, other genes related to LOX activation and matrix production were upregulated. For the loading study, the combined effects of BAPN and mechanical loading were assessed. Gene expression was quantified, atomic force microscopy was used to extract elastic properties of the collagen matrix, and Fourier Transform infrared spectroscopy was used to assess collagen secondary structure for enzymatic crosslinking analysis. BAPN upregulated BMP-1 in static samples and BAPN combined with mechanical loading downregulated LOX when compared to control-static samples. Results showed a higher indentation modulus in BAPN-loaded samples compared to control-loaded samples. Loading increased the mature-to-immature crosslink ratios in control samples, and BAPN increased the height ratio in static samples. In summary, effects of BAPN (upregulation of genes involved in crosslinking, mature/immature crosslinking ratios, upward trend in collagen elasticity) were mitigated by mechanical loading.

Inhibition of enzymes involved in collagen cross-linking reduces vascular smooth muscle cell calcification.

Vascular smooth muscle cells (VSMCs) transdifferentiate into osteoblast-like cells during vascular calcification, inducing active remodeling and calcification of the extracellular matrix (ECM). Intracellular and extracellular enzymes, such as lysyl hydroxylase 1 (PLOD1) and lysyl oxidase (LOX), contribute to ECM maturation and stabilization. We assessed the contribution of these enzymes to hyperphosphatemia-induced calcification. Human and murine VSMCs were differentiated into functional osteoblast-like cells by high-phosphate medium (HPM) conditioning. HPM promoted ECM calcification and up-regulated osteoblast markers associated with induction of LOX and PLOD1 expression and with an increase in ECM-insoluble collagen deposition. Murine VSMCs from transgenic mice overexpressing LOX (TgLOX) exhibited an increase in HPM-dependent calcification and osteoblast commitment compared with wild-type cells. Similarly, enhanced HPM-induced calcification was detected in aorta from TgLOX. Conversely, ß-aminopropionitrile (a LOX inhibitor) and LOX knockdown abrogated VSMC calcification and transdifferentiation. We found a significant positive association between LOX expression and vascular calcification in human atherosclerotic lesions. Likewise, 2,2'-dipyridil (a PLOD inhibitor) and PLOD1 knockdown impaired HPM-induced ECM mineralization and osteoblast commitment. Our findings identify LOX and PLOD as critical players in vascular calcification and highlight the importance of ECM remodeling in this process.-Jover, E., Silvente, A., Marín, F., Martínez-González, J., Orriols, M., Martinez, C. M., Puche, C. M., Valdés, M., Rodriguez, C., Hernández-Romero, D. Inhibition of enzymes involved in collagen cross-linking reduces vascular smooth muscle cell calcification.

In vivo cerebral aneurysm models.

Cerebral aneurysm rupture is a devastating event resulting in subarachnoid hemorrhage and is associated with significant morbidity and death. Up to 50% of individuals do not survive aneurysm rupture, with the majority of survivors suffering some degree of neurological deficit. Therefore, prior to aneurysm rupture, a large number of diagnosed patients are treated either microsurgically via clipping or endovascularly to prevent aneurysm filling. With the advancement of endovascular surgical techniques and devices, endovascular treatment of cerebral aneurysms is becoming the first-line therapy at many hospitals. Despite this fact, a large number of endovascularly treated patients will have aneurysm recanalization and progression and will require retreatment. The lack of approved pharmacological interventions for cerebral aneurysms and the need for retreatment have led to a growing interest in understanding the molecular, cellular, and physiological determinants of cerebral aneurysm pathogenesis, maturation, and rupture. To this end, the use of animal cerebral aneurysm models has contributed significantly to our current understanding of cerebral aneurysm biology and to the development of and training in endovascular devices. This review summarizes the small and large animal models of cerebral aneurysm that are being used to explore the pathophysiology of cerebral aneurysms, as well as the development of novel endovascular devices for aneurysm treatment.

A Lox/CHOP-10 crosstalk governs osteogenic and adipogenic cell fate by MSCs.

Accelerated marrow adipogenesis has been associated with ageing and osteoporosis and is thought to be because of an imbalance between adipogenic and osteogenic differentiation of mesenchymal stem cell (MSCs). We have previously found that lysyl oxidase (Lox) inhibition disrupts BMP4-induced adipocytic lineage commitment and differentiation of MSCs. In this study, we found that lox inhibition dramatically up-regulates BMP4-induced expression of CCAAT/enhancer binding protein (C/EBP) homologous protein 10 (CHOP-10), which then promotes BMP4-induced osteogenesis of MSCs both in vitro and in vivo. Specifically, Lox inhibition or CHOP-10 up-regulation activated Wnt/ß-catenin signalling to enhance BMP4-induced osteogenesis, with pro-adipogenic p38 MAPK and Smad signalling suppressed. Together, we demonstrate that Lox/CHOP-10 crosstalk regulates BMP4-induced osteogenic and adipogenic fate determination of MSCs, presenting a promising therapeutic target for osteoporosis and other bone diseases.

The Effect of ß-Aminopropionitrile on Skeletal Micromorphology and Osteogenesis.

Collagen cross-linking, as a form of collagen post-translational modification, plays a crucial role in maintaining bone mechanical properties as well as in regulating cell biological functions. Shifts in cross-links profile are found apparently correlated to kinds of skeletal pathology and diseases, whereas little is known about the relationship between collagen cross-links and osteogenesis. Here, we hypothesized that the inhibition of collagen cross-links could impair skeletal microstructure and inhibit osteogenesis. A mouse model of collagen cross-linking defects has been established using subcutaneous injection of 350 mg/kg ß-aminopropionitrile (BAPN) daily for 4 weeks, and same dose of phosphate buffered saline (PBS) served as control group. The analysis of bone microstructural parameters revealed a significant decrease of bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), and increase of bone surface ratio (BS/BV), structure model index (SMI) as well as trabecular separation (Tb.Sp) in the experimental group (p < 0.05), whereas there was no difference observed in bone mineral density (BMD). Histological staining displayed that the BAPN treatment caused thinner trabeculae and decrease of collagen content in proximal tibiae. The analysis of osteogenesis PCR (Polymerase Chain Reaction) array reflected that BAPN remarkably influenced the expression of Alpl, Bglap, Bgn, Bmp5, Col10a1, Col1a1, Col1a2, Col5a1, Itga2b, and Serpinh1. The results of immunohistochemistry displayed a significant reduction in the mean optical densities of OCN and COL1 at the presence of BAPN. The overall results of this study suggested that BAPN alters bone microstructure and hinders the expression of osteogenic genes without affecting mineralization processes, indicating the influences of collagen cross-links on osteogenesis may be a potential pathological mechanism in skeletal diseases.

Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

Collagen stabilization through irreversible cross-linking is thought to promote hepatic fibrosis progression and limit its reversibility. However, the mechanism of this process remains poorly defined. We studied the functional contribution of lysyl oxidase (LOX) to collagen stabilization and hepatic fibrosis progression/reversalin vivousing chronic administration of irreversible LOX inhibitor ß-aminopropionitrile (BAPN, or vehicle as control) in C57Bl/6J mice with carbon tetrachloride (CCl4)-induced fibrosis. Fibrotic matrix stability was directly assessed using a stepwise collagen extraction assay and fibrotic septae morphometry. Liver cells and fibrosis were studied by histologic, biochemical methods and quantitative real-time reverse-transcription PCR. During fibrosis progression, BAPN administration suppressed accumulation of cross-linked collagens, and fibrotic septae showed widening and collagen fibrils splitting, reminiscent of remodeling signs observed during fibrosis reversal. LOX inhibition attenuated hepatic stellate cell activation markers and promoted F4/80-positive scar-associated macrophage infiltration without an increase in liver injury. In reversal experiments, BAPN-treated fibrotic mice demonstrated accelerated fibrosis reversal after CCl4withdrawal. Our findings demonstrate for the first time that LOX contributes significantly to collagen stabilization in liver fibrosis, promotes fibrogenic activation of attenuated hepatic stellate cells, and limits fibrosis reversal. Our data support the concept of pharmacologic targeting of LOX pathway to inhibit liver fibrosis and promote its resolution.-Liu, S. B., Ikenaga, N., Peng, Z.-W., Sverdlov, D. Y., Greenstein, A., Smith, V., Schuppan, D., Popov, Y. Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice.

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells.

Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used ß-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation.

Quantification of ß-aminopropionitrile, an inhibitor of lysyl oxidase activity, in plasma and tumor of mice by liquid chromatography tandem mass spectrometry.

Lysyl oxidase enzymes are reported to be involved in patho-physiological process such as tumorigenesis. ß-Aminopropionitrile (BAPN) is an irreversible inhibitor of lysyl oxidase activity, suggesting a potentially useful therapeutic of interest in oncology. This paper describes the first assay concerning the quantification of BAPN by mass spectrometry. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed for the quantification of BAPN in plasma and tumor of mice. This method combines dansyl chloride (Dns) derivatization and extraction using a solid-phase extraction Oasis Max column. Deuterated BAPN was used as internal standard (IS). Separation was achieved using an C18 column HypersylGold, (ThermoElectron), 3.0 µm (100 × 2.1 mm i.d.). Gradient elution with water containing 0.1% acetic acid (A) and acetonitrile containing 0.1% acetic acid (B) was applied. Detection was performed with an electrospray ionization interface operating in negative ion mode. Selected reaction monitoring was used with ion transitions m/z 302 → 249 for BAPN-Dns and m/z 306 → 250 for the IS. The method was fully validated in plasma and was linear and sensitive in the range of 10-500 ng/mL. The lower limit of quantification in plasma was 2.5 ng/mL. This validated assay was successfully applied to a kinetic study of BAPN in mouse plasma and demonstrates that BAPN reaches the tumoral tissue.

Inducing aortic aneurysm/dissection in zebrafish: evaluating the efficacy of ß-Aminopropionic Nitrile as a model.

Aortic aneurysm/dissection (AAD) poses a life-threatening cardiovascular emergency with complex mechanisms and a notably high mortality rate. Zebrafish (Danio rerio) serve as valuable models for AAD due to the conservation of their three-layered arterial structure and genome with that of humans. However, the existing studies have predominantly focused on larval zebrafish, leaving a gap in our understanding of adult zebrafish. In this study, we utilized ß-Aminopropionic Nitrile (BAPN) impregnation to induce AAD in both larval and adult zebrafish. Following induction, larval zebrafish exhibited a 28% widening of the dorsal aortic diameter (p < 0.0004, n = 10) and aortic arch malformations, with a high malformation rate of 75% (6/8). Conversely, adult zebrafish showed a 41.67% (5/12) mortality rate 22 days post-induction. At this time point, the dorsal aortic area had expanded by 2.46 times (p < 0.009), and the vessel wall demonstrated significant thickening (8.22 ± 2.23 µM vs. 26.38 ± 10.74 µM, p < 0.05). Pathological analysis revealed disruptions in the smooth muscle layer, contributing to a 58.33% aneurysm rate. Moreover, the expression levels of acta2, tagln, cnn1a, and cnn1b were decreased, indicating a weakened contractile phenotype. Transcriptome sequencing showed a significant overlap between the molecular features of zebrafish tissues post-BAPN treatment and those of AAD patients. Our findings present a straightforward and practical method for generating AAD models in both larval and adult zebrafish using BAPN.

Modulation of arterial intima stiffness by disturbed blood flow.

The intima, comprising the endothelium and the subendothelial matrix, plays a crucial role in atherosclerosis pathogenesis. The mechanical stress arising from disturbed blood flow (d-flow) and the stiffening of the arterial wall contributes to endothelial dysfunction. However, the specific impacts of these physical forces on the mechanical environment of the intima remain undetermined. Here, we investigated whether inhibiting collagen crosslinking could ameliorate the detrimental effects of persistent d-flow on the mechanical properties of the intima. Partial ligation of the left carotid artery (LCA) was performed in C57BL/6J mice, inducing d-flow. The right carotid artery (RCA) served as an internal control. Carotids were collected 2 days and 2 weeks after surgery to study acute and chronic effects of d-flow on the mechanical phenotype of the intima. The chronic effects of d-flow were decoupled from the ensuing arterial wall stiffening by administration of ß-aminopropionitrile (BAPN), an inhibitor of collagen crosslinking by lysyl oxidase (LOX) enzymes. Atomic force microscopy (AFM) was used to determine stiffness of the endothelium and the denuded subendothelial matrix in en face carotid preparations. The stiffness of human aortic endothelial cells (HAEC) cultured on soft and stiff hydrogels was also determined. Acute exposure to d-flow caused a slight decrease in endothelial stiffness in male mice but had no effect on the stiffness of the subendothelial matrix in either sex. Regardless of sex, the intact endothelium was softer than the subendothelial matrix. In contrast, exposure to chronic d-flow led to a substantial increase in the endothelial and subendothelial stiffness in both sexes. The effects of chronic d-flow were largely prevented by concurrent BAPN administration. In addition, HAEC displayed reduced stiffness when cultured on soft vs. stiff hydrogels. We conclude that chronic d-flow results in marked stiffening of the arterial intima, which can be effectively prevented by inhibition of collagen crosslinking.

Delamination Strength and Elastin Interlaminar Fibers Decrease with the Development of Aortic Dissection in Model Rats.

Aortic dissection (AD) is a life-threatening tear of the vascular tissue with creation of a false lumen. To explore the mechanism underlying this tissue tear, this study investigated the delamination strength of AD model rats and the histological composition of the aorta at various stages of AD development. SD rats were administrated beta-amino propionitrile for 0 (Control), 3 (Pre-dissection), and 6 (Dissection) weeks. The thoracic aorta was harvested at 10-11 weeks of age. The Dissection group exclusively showed AD at the ascending aorta. The delamination strength, a force that separates the aorta in the radial direction, of the descending aorta decreased significantly in the order of the Control, Pre-dissection, and Dissection groups. A quantitative histological analysis of the aortic tissue demonstrated that, compared with the Control group, the area fraction of collagen was significantly higher in the Pre-dissection and Dissection groups and that of elastin was significantly lower in the Dissection group. The area fraction of the elastin fibers between the elastic laminas (interlaminar fibers) was significantly decreased in the order of the Control, Pre-dissection, and Dissection groups. Histological changes of the aortic tissue, perhaps a reduction in interlaminar fibers mainly aligned in the radial direction, decreased delamination strength, thereby causing AD.

ADAMTS-7 deficiency attenuates thoracic aortic aneurysm and dissection in mice.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening cardiovascular disease with severe extracellular matrix (ECM) remodeling that lacks efficient early stage diagnosis and nonsurgical therapy. A disintegrin and metalloproteinase with thrombospondin motif 7 (ADAMTS-7) is recognized as a novel locus for human coronary artery atherosclerosis. Previous work by us and others showed that ADAMTS-7 promoted atherosclerosis, postinjury neointima formation, and vascular calcification. However, whether ADAMTS-7 is involved in TAAD pathogenesis is unknown. We aimed to explore the alterations in ADAMTS-7 expression in human and mouse TAAD, and investigate the role of ADAMTS-7 in TAAD formation. A case-control study of TAAD patients (N = 86) and healthy participants (N = 88) was performed. The plasma ADAMTS-7 levels were markedly increased in TAAD patients within 24 h and peaked in 7 days. A TAAD mouse model was induced with 0.5% ß-aminopropionitrile (BAPN) in drinking water. ELISA analysis of mouse plasma, Western blotting, and immunohistochemical staining of aorta showed an increase in ADAMTS-7 in the early stage of TAAD. Moreover, ADAMTS-7-deficient mice exhibited significantly attenuated TAAD formation and TAAD rupture-related mortality in both male and female mice, which was accompanied by reduced artery dilation and inhibited elastin degradation. ADAMTS-7 deficiency caused repressed inflammatory response and complement system activation during TAAD formation. An increase in plasma ADAMTS-7 is a novel biomarker for human TAAD. ADAMTS-7 deficiency attenuates BAPN-induced murine TAAD. ADAMTS-7 is a potential novel target for TAAD diagnosis and therapy. KEY MESSAGES: A case-control study revealed increased plasma ADAMTS-7 is a risk factor for TAAD. ADAMTS-7 was elevated in plasma and aorta at early stage of mouse TAAD. ADAMTS-7 knockout attenuated mouse TAAD formation and mortality in both sexes.

Comparative bioinformatics analysis of transcriptomes between ß-aminopropionitrile-induced aortic dissection murine model and human aortic dissection.

Background: Aortic dissection (AD) poses a great threat to the life of patients; however, there is currently no documentation of a clear pathogenic mechanism of this disease. In recent years, ß-aminopropionitrile (BAPN)-induced AD in rodents has been widely used in basic research, which provides a good platform for exploring the pathogenesis of AD and drug modification. This study aimed to identify molecular markers and pathways for the diagnosis and treatment of AD by comparing a murine AD model and human AD transcriptome through a bioinformatics analysis. Methods: We constructed a BAPN-induced mice model and performed high-throughput sequencing analysis. The GSE147026 dataset of patients was obtained from the Gene Expression Omnibus database. We performed a subsequent bioinformatics analysis of human AD and the murine AD model using R software. The DESeq software package was used to analyze the differentially expressed genes (DEGs). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to analyze the enrichment pathways. Protein-protein interaction network construction and hub gene selection were based on STRING software analysis. Stepwise identification of potential drugs was performed online, while hub genes were validated immunohistochemically. Results: We compared the murine AD model and human AD transcriptome and found that both differentially expressed 463 genes. The cytokine-cytokine receptor interaction, tuberculosis, and phagosome pathways were significantly enriched. CDC20, CCNB2, and CCNB1 may be associated with AD development. Protein-drug interactions were also identified. Conclusions: This study is the first to reveal transcriptional changes in a murine BAPN-induced AD model versus human AD transcriptome. Furthermore, we identified the important hub genes, related pathways, and potential drugs by analyzing the overlapping DEGs between human AD and the murine AD model. Our results provide a basis for the further identification of potential molecular markers for diagnosing and treating AD.

Mechanical and matrix effects of short and long-duration exposure to beta-aminopropionitrile in elastase-induced model abdominal aortic aneurysm in mice.

Objective: Evaluate the mechanical and matrix effects on abdominal aortic aneurysms (AAA) during the initial aortic dilation and after prolonged exposure to beta-aminopropionitrile (BAPN) in a topical elastase AAA model. Methods: Abdominal aortae of C57/BL6 mice were exposed to topical elastase with or without BAPN in the drinking water starting 4 days before elastase exposure. For the standard AAA model, animals were harvested at 2 weeks after active elastase (STD2) or heat-inactivated elastase (SHAM2). For the enhanced elastase model, BAPN treatment continued for either 4 days (ENH2b) or until harvest (ENH2) at 2 weeks; BAPN was continued until harvest at 8 weeks in one group (ENH8). Each group underwent assessment of aortic diameter, mechanical testing (tangent modulus and ultimate tensile strength [UTS]), and quantification of insoluble elastin and bulk collagen in both the elastase exposed aorta as well as the descending thoracic aorta. Results: BAPN treatment did not increase aortic dilation compared with the standard model after 2 weeks (ENH2, 1.65 ± 0.23 mm; ENH2b, 1.49 ± 0.39 mm; STD2, 1.67 ± 0.29 mm; and SHAM2, 0.73 ± 0.10 mm), but did result in increased dilation after 8 weeks (4.3 ± 2.0 mm; P = .005). After 2 weeks, compared with the standard model, continuous therapy with BAPN did not have an effect on UTS (24.84 ± 7.62 N/cm2; 18.05 ± 4.95 N/cm2), tangent modulus (32.60 ± 9.83 N/cm2; 26.13 ± 9.10 N/cm2), elastin (7.41 ± 2.43%; 7.37 ± 4.00%), or collagen (4.25 ± 0.79%; 5.86 ± 1.19%) content. The brief treatment, EHN2b, resulted in increased aortic collagen content compared with STD2 (7.55 ± 2.48%; P = .006) and an increase in UTS compared with ENH2 (35.18 ± 18.60 N/cm2; P = .03). The ENH8 group had the lowest tangent modulus (3.71 ± 3.10 N/cm2; P = .005) compared with all aortas harvested at 2 weeks and a lower UTS (2.18 ± 2.18 N/cm2) compared with both the STD2 (24.84 ± 7.62 N/cm2; P = .008) and ENH2b (35.18 ± 18.60 N/cm2; P = .001) groups. No differences in the mechanical properties or matrix protein concentrations were associated with abdominal elastase exposure or BAPN treatment for the thoracic aorta. The tangent modulus was higher in the STD2 group (32.60 ± 9.83 N/cm2; P = .0456) vs the SHAM2 group (17.99 ± 5.76 N/cm2), and the UTS was lower in the ENH2 group (18.05 ± 4.95 N/cm2; P = .0292) compared with the ENH2b group (35.18 ± 18.60 N/cm2). The ENH8 group had the lowest tangent modulus (3.71 ± 3.10 N/cm2; P = .005) compared with all aortas harvested at 2 weeks and a lower UTS (2.18 ± 2.18 N/cm2) compared with both the STD2 (24.84 ± 7.62 N/cm2; P = .008) and ENH2b (35.18 ± 18.60 N/cm2; P = .001) groups. Abdominal aortic elastin in the STD2 group (7.41 ± 2.43%; P = .035) was lower compared with the SHAM2 group (15.29 ± 7.66%). Aortic collagen was lower in the STD2 group (4.25 ± 0.79%; P = .007) compared with the SHAM2 group (12.44 ± 6.02%) and higher for the ENH2b (7.55 ± 2.48%; P = .006) compared with the STD2 group. Conclusions: Enhancing an elastase AAA model with BAPN does not affect the initial (2-week) dilation phase substantially, either mechanically or by altering the matrix content. Late mechanical and matrix effects of prolonged BAPN treatment are limited to the elastase-exposed segment of the aorta. Clinical Relevance: This paper explores the use of short- and long-term exposure to beta-aminopropionitrile to create an enhanced topical elastase abdominal aortic aneurysm model in mice. Readouts of aneurysm severity included loss of mechanical stability and vascular extracellular matrix composition reminiscent of what is seen in the course of human disease. Additionally, we show that the thoracic aorta, unlike the findings below the renal arteries, is not damaged in our animal model.

Microenergy acoustic pulse therapy restores function and structure of pelvic floor muscles after simulated birth injury.

Background: The mechanisms of the microenergy acoustic pulse (MAP) therapy on restoring structure and function of pelvic floor muscles (PFM) after simulated birth injury are not well understood. Methods: A total 24 female Sprague-Dawley rats were randomly grouped into sham control (sham), vaginal balloon dilation and ovariectomy (VBDO), VBDO + ß-aminopropionitrile (BAPN, an irreversible LOX inhibitor), and VBDO + BAPN and treated with MAP (n=6 in each group). The MAP therapy was administered 2 times per week for 4 weeks with 1-week washout, the functional and histological studies were conducted in all 24 rats. The viscoelastic behavior of the PFM, including iliococcygeus (IC) and pubococcygeus (PC), was examined with a biomechanical assay. The structure of the PFM was assessed by immunofluorescence and Masson's trichrome staining. Results: The leak point pressure (LPP) assay demonstrated that the MAP therapy group had higher LPPs compared to that of VBDO and BAPN groups. In the sham group, the muscular stiffness (K) of IC muscle was significantly higher than that of PC muscle while the pelvic floor muscle rebound activity (MRA) of PC muscle was stronger than that of IC muscle (291.26±45.33 and 241.18±14.23 N/cm2, respectively). Both VBDO and BAPN decreased the MRA and increased the K in both IC and PC. Histologic examination revealed increased fibrous tissue (collagen) and degeneration of muscle fibers in both VBDO and BAPN groups. MAP therapy significantly reduced the collagen content and improved the architecture of muscle fibers. Conclusions: MAP appears to restore the structure and function of PFM by regenerating muscular fibers and improving biomechanical properties in an animal model of simulated birth injury.