Inhibition of heme oxygenase 1 alleviates thoracic aortic aneurysm via restoration of extracellular matrix.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a silent but life-threatening cardiovascular disease. Heme oxygenase 1 (HO-1) plays an important role in the cardiovascular diseases but is poorly understood in TAA. This study aims at investigating the role of HO-1 in TAA. METHODS: Single-cell RNA sequencing, Western blot and histological assay were performed to identify specific cellular expression of HO-1 in both human and ß-aminopropionitrile (BAPN)-induced mice TAA. Zinc protoporphyrin (ZnPP), a pharmacological inhibitor of HO-1, was used to investigate whether inhibition of HO-1 could attenuate BAPN-induced TAA in rodent model. Histological assay, Western blot assay, and mRNA sequencing were further performed to explore the underlying mechanisms. RESULTS: Single-cell transcriptomic analyses of 113,800 thoracic aortic cells identified an increase of HO-1(+) macrophage in aneurysmal thoracic aorta from BAPN-induced TAA mice and TAA patients. Histological assay verified HO-1 overexpression in clinical TAA specimens, which was co-localized with CD68(+) macrophage. HO-1(+) macrophage was closely associated with pro-inflammatory response and immune activation. Inhibition of HO-1 through ZnPP significantly alleviated BAPN-induced TAA in mice and restored extracellular matrix (ECM) in vivo. Further experiments showed that ZnPP treatment suppressed the expression of matrix metalloproteinases (MMPs) in aneurysmal thoracic aortic tissues from BAPN-induced TAA mice, including MMP2 and MMP9. Macrophages from myeloid specific HO-1 knockout mice displayed weakened pro-inflammatory activity and ECM degradation capability. CONCLUSION: HO-1(+) macrophage subgroup is a typical hallmark of TAA. Inhibition of HO-1 through ZnPP alleviates BAPN-induced TAA in mice, which might work through restoration of ECM via suppressing MMP2 and MMP9 expression.

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.

Intramural injection of pluronic gel loaded with drugs to alleviate arterial injury.

BACKGROUND: Balloon angioplasty, stent implantation, and application of an arterial clamp during surgery can induce artery injury such as elastin breaks and endothelium injury, but there is little research focused on the injury induced by these therapeutic manipulations. We established a simple and reproducible small animal aortic injury model and examined intramural injection as a potential therapeutic method to alleviate injury. MATERIALS AND METHODS: The abdominal aorta of male Sprague Dawley (SD) rats or C57BL/6 J mice was clamped sequentially throughout its length. Transforming growth factor ß1 (TGFß1), SB431542, lipopolysaccharide (LPS), Necrostatin-1 (Nec-1), rapamycin, or MHY1485 contained in Pluronic gel was injected intramurally at day 0 or day 7. Animals were fed with chow containing 0.25% beta-aminopropionitrile (BAPN) to evaluate the influence of BAPN. All samples were harvested and examined by immunohistochemistry and immunofluorescence. RESULTS: The clamped rat aorta showed luminal dilation, elastin fiber breaks, neointimal hyperplasia, and dissection (days 0-90). Intramural injection of TGFß1, rapamycin and Nec-1 showed a protective effect on the injured aorta, whereas SB431542, MHY1485 and LPS showed more severe wall damage. The aortic lumen in rats fed with BAPN was significantly larger than in control rats (day 7). Mouse aorta showed similar injury with neointimal hyperplasia and elastin fiber breaks. CONCLUSIONS: The rodent arterial injury model is reproducible and may mimic early changes of arterial injury. The model accommodates intramural injection of different drugs that may show mechanisms of arterial injury. Although this is a preliminary animal model, the intramural injection method may have potential clinical application in the future.

Dexamethasone reduces the formation of thoracic aortic aneurysm and dissection in a murine model.

Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening vascular disease with no effective pharmaceutical therapies currently available. Inflammation plays a key role in the progression of aneurysms. Dexamethasone (DEX), a synthetic glucocorticoid, has showed alleviating effects on cells in vitro from TAAD patients. Here we performed a study aiming at investigating the protective role of DEX in a ß-aminopropionitrile monofumarate (BAPN)-induced TAAD mouse model. DEX (dose: 0.04 mg/kg/day) treatment significantly reduced the aortic diameter and inhibited TAAD formation. DEX reduced infiltration of macrophages and neutrophils, apoptosis of vascular smooth muscle cells (VSMCs), expression of metalloproteinase 2/9, and extracellular matrix degradation in BAPN-treated TAAD mice. Furthermore, DEX therapy downregulated the expression of p-p65 in macrophages and VSMCs, which suggested that DEX might ameliorate BAPN-induced TAAD by suppressing NF-κB signaling. Therefore, DEX therapy attenuates the progression of BAPN-induced TAAD murine model and could be used as an effective adjuvant therapy for treating TAAD.

Arterial stiffness and cardiac dysfunction in Hutchinson-Gilford Progeria Syndrome corrected by inhibition of lysyl oxidase.

Arterial stiffening and cardiac dysfunction are hallmarks of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), but the molecular regulators remain unknown. Here, we show that the LaminAG609G mouse model of HGPS recapitulates the premature arterial stiffening and early diastolic dysfunction seen in human HGPS. Lysyl oxidase (LOX) is up-regulated in the arteries of these mice, and treatment with the LOX inhibitor, ß-aminopropionitrile, improves arterial mechanics and cardiac function. Genome-wide and mechanistic analysis revealed reduced expression of the LOX-regulator, miR-145, in HGPS arteries, and forced expression of miR-145 restores normal LOX gene expression in HGPS smooth muscle cells. LOX abundance is also increased in the carotid arteries of aged wild-type mice, but its spatial expression differs from HGPS and its up-regulation is independent of changes in miR-145 abundance. Our results show that miR-145 is selectively misregulated in HGPS and that the consequent up-regulation of LOX is causal for premature arterial stiffening and cardiac dysfunction.

Bilateral superior cervical ganglionectomy attenuates the progression of ß-aminopropionitrile-induced aortic dissection in rats.

AIMS: Aortic dissection (AD) represents one of the most common aortic emergencies with high incidence of morbidity and mortality. Clinical studies have shown that the increased excitability of the sympathetic nerve may be associated with the formation of AD. In this study, we examined the effects of bilateral superior cervical sympathectomy (SCGx) on the progression of ß-aminopropionitrile (BAPN)-induced AD in rats. MAIN METHODS: Sprague-Dawley rats were randomly divided into three groups, including BAPN, BAPN+SCGx and control groups. For terminal measurements, the mean arterial pressure (MAP) and heart rate (HR) were monitored and the basal sympathetic nerve activity (SNA) was assessed through recording the variation in arterial pressure in response to hexamethonium application. Pathological changes in the aortic wall were observed by histological staining. Matrix metalloproteinase-2 (MMP-2) and MMP-9 concentrations within the aortic wall were analyzed by western blot. KEY FINDINGS: The results show that BAPN administration could elevate SNA and cause the formation of AD in rats with a high incidence (67.7%), while SCGx treatment inhibited the elevation of SNA and significantly reduced the incidence (20%). SCGx may suppress the formation of BAPN-induced AD via restraining the rise of HR and reducing the MMP-9 concentration in aortic wall. SIGNIFICANCE: These results indicate that surgical techniques such as sympathetic nerve block may be a potentially useful therapy for the prevention of AD.

Vascular ADAM17 (a Disintegrin and Metalloproteinase Domain 17) Is Required for Angiotensin II/ß-Aminopropionitrile-Induced Abdominal Aortic Aneurysm.

Angiotensin II (AngII)-activated epidermal growth factor receptor has been implicated in abdominal aortic aneurysm (AAA) development. In vascular smooth muscle cells (VSMCs), AngII activates epidermal growth factor receptor via a metalloproteinase, ADAM17 (a disintegrin and metalloproteinase domain 17). We hypothesized that AngII-dependent AAA development would be prevented in mice lacking ADAM17 in VSMCs. To test this concept, control and VSMC ADAM17-deficient mice were cotreated with AngII and a lysyl oxidase inhibitor, ß-aminopropionitrile, to induce AAA. We found that 52.4% of control mice did not survive because of aortic rupture. All other surviving control mice developed AAA and demonstrated enhanced expression of ADAM17 in the AAA lesions. In contrast, all AngII and ß-aminopropionitrile-treated VSMC ADAM17-deficient mice survived and showed reduction in external/internal diameters (51%/28%, respectively). VSMC ADAM17 deficiency was associated with lack of epidermal growth factor receptor activation, interleukin-6 induction, endoplasmic reticulum/oxidative stress, and matrix deposition in the abdominal aorta of treated mice. However, both VSMC ADAM17-deficient and control mice treated with AngII and ß-aminopropionitrile developed comparable levels of hypertension. Treatment of C57Bl/6 mice with an ADAM17 inhibitory antibody but not with control IgG also prevented AAA development. In conclusion, VSMC ADAM17 silencing or systemic ADAM17 inhibition seems to protect mice from AAA formation. The mechanism seems to involve suppression of epidermal growth factor receptor activation.

Effect of cellular aging on collagen biosynthesis: I. Methodological considerations and pharmacological applications.

The study of the age and passage dependent modifications of collagen biosynthesis requires a simple, rapid and reproducible procedure adaptable to serial cell cultures. To make such a method comparable to other methods of collagen determination, we calibrated a colorimetric procedure both by hydroxyproline (HYP) determinations and in terms of collagen concentration. For collagen types I and IV, widely different slopes were obtained with the colorimetric procedure. To further refine the procedure, we tempted to completely inhibit collagen synthesis by beta-aminopropionitrile (beta APN) added to cultures in order to obtain a negative control. Using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl 2H-tetrazolium bromide (MTT-test), it could be shown that relatively high concentrations of beta APN are tolerated by the cells. It appeared, however, that even the highest concentration of beta APN (1mM) still tolerated by the fibroblasts did not completely inhibit collagen synthesis. At low concentrations, beta APN even stimulated cell-proliferation. The colorimetric procedure calibrated in terms of collagen type I concentration, was therefore retained for the serial determination of collagen synthesis and accumulation. We shall here describe the methodological details of its validation as well as its application for the pharmacological study of the effect of aging on collagen biosynthesis. Among the factors involved, the accumulation of advanced glycation end-products (AGEs) might well play an important role. Several of such AGE-products showed a significant inhibition of collagen deposition. On the contrary, retinol, ascorbic acid as well as the rhamnose-rich oligo- and polysaccharides (RROPs) did produce a significant upregulation collagen deposition. Polysaccharide preparations, rich in rhamnose and fucose (the EROB-mixture) could protect against the AGEs-induced inhibition of collagen accumulation.

Kinetics of collagen crosslinking in adult bovine articular cartilage.

OBJECTIVE: Determine the kinetics of collagen crosslinking in adult bovine articular cartilage explants using radiolabel pulse-chase studies. METHODS: Explant cultures of adult bovine articular cartilage were radiolabeled with [14C]lysine in medium including fetal bovine serum and ascorbate, and then maintained for chase periods up to 28 days. In some samples, beta-aminopropionitrile (BAPN) was included during chase to inhibit lysyl oxidase-mediated collagen crosslinking. Tissue was hydrolyzed and analyzed for [14C]metabolites in the forms of lysine, hydroxylysine, dehydrodihydroxylysinonorleucine (DeltaDHLNL), and hydroxylysyl pyridinoline (HP). RESULTS: Explant cultures of adult bovine articular cartilage metabolized lysine into hydroxylysine and the collagen crosslinks, DeltaDHLNL and HP. During chase, [14C]hydroxylysine maintained steady-state levels, [14C]DHLNL rose to a plateau, and [14C]HP increased gradually. Addition of BAPN inhibited formation of [14C]DHLNL. Analysis of raw data and that normalized to [14C]hydroxylysine gave characteristic time constants for formation of DeltaDHLNL and HP crosslinks of 1-2 and 7-30 days, respectively. The distribution of [14C]lysine metabolites in collagen crosslinks was described by peak values in [14C]DHLNL/[14C]hydroxylysine of 0.047-0.064 and in [14C]HP/[14C]hydroxylysine of 0.03. CONCLUSION: Collagen crosslinks form in cartilage explants in vitro according to the classical lysyl oxidase-mediated pathway.

Role of monoamine oxidase in aminopropionitrile-induced neurotoxicity.

Oxidation of aminopropionitriles was measured in vitro with both rat liver mitochondria and bovine plasma monoamine oxidase (MAO). The nonneurotoxic aminonitrile beta-aminopropionitrile (BAPN) was oxidized at a significantly higher rate (p less than .05) than either of the neurotoxic aminonitriles tested; 3,3'-iminodipropionitrile (IDPN) and 3,3'-dimethylaminopropionitrile (DMAPN). DMAPN was a poor substrate for both mitochondrial and plasma MAO. None of the aminonitriles tested were found to inhibit MAO activity in rat brain or liver in vivo. Inhibition of MAO activity with pargyline in vivo did not affect the pattern of IDPN- or DMAPN-induced toxicity. These results suggest that monoamine oxidase is not involved in aminonitrile-induced neurotoxicity.

Induction of lung lysyl oxidase activity and lysyl oxidase protein by exposure of rats to cadmium chloride: properties of the induced enzyme.

Inspiration of CdCl2 results in a focally fibrotic response in rat lungs and markedly increases the activity of lung lysyl oxidase. Western blot analyses of urea-extractable rat lung proteins revealed that the levels of an immunoreactive, 32,000-Da protein were markedly increased in the cadmium-exposed rat lung tissue, consistent with the induction of lysyl oxidase protein. Anion exchange chromatography revealed low levels of multiple peaks of catalytically functional lysyl oxidase in control rat lung extracts, while the profile of cadmium-exposed rat lung extracts displayed markedly elevated levels of multiple peaks of enzyme activity indicating that the charge heterogeneity is expressed in the activated enzyme. The cadmium-induced enzyme was purified as a species of 32 kDa, without resolving individual ionic variants. The catalytic and physical properties of the isolated enzyme were very similar to those of previously well characterized basal enzyme of bovine aorta, including the presence of a pyrroloquinoline quinone-like carbonyl cofactor. The copper and cadmium content of the cadmium-induced enzyme indicated little if any replacement of tightly-bound copper by cadmium in the exposed lung.

[Production of cyanide in the rat liver from 3-aminopropionitrile]. / Production de cyanure dans le foie de rat à partir de 3-aminopropionitrile.

CN- Production was investigated from a saturated mononitrile, 3-aminopropionitrile, in small liver pieces and liver homogenates. Results show that CN- production is a function of time and of the concentration of active ingredient, with a trend to saturation in both cases. Similarly, cell integrity is not required for the biotransformation of 3-aminopropionitrile to occur. Cofactors such as NADPH must be present to reach high level activities. The effect of free radicals scavengers such as mannitol and DMSO was also studied.

An ultrafiltration assay for lysyl oxidase.

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.

Reaction of pig plasma benzylamine oxidase with beta-aminopropionitrile.

Beta-aminopropionitrile (BAPN) is an inhibitor of pig plasma benzylamine oxidase. BAPN is oxidized by benzylamine oxidase. Inhibition develops in a time-dependent fashion upon incubation of BAPN with the enzyme in the absence of substrate. The product of oxidation of BAPN by benzylamine oxidase, cyanacetaldehyde, was identified and prepared by synthesis. It is an irreversible inhibitor of the enzyme.

Evaluation of the inhibition of glycolytic enzymes by the neurotoxicant dimethylaminopropionitrile.

The neurotoxic aminonitrile dimethylaminopropionitrile (DMAPN) inhibits the crystalline glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-6-phosphate kinase (phosphofructokinase, PFK). Preincubation of GAPDH with the aminonitrile enhances the inhibition, indicating that the inhibition is irreversible. The overall bimolecular rate constant ki was determined to be 2.42 +/- 0.21 M-1 min-1. Dithiothreitol (DTT) partially protected the enzyme from inhibition. PFK is also inhibited by DMAPN, but the inhibition is reversible and noncompetitive with a Kl, of 2.79 X 10(-2) M. DMAPN does not inhibit lactate dehydrogenase (LDH), nor is GAPDH or PFK inhibited by 3,3'-iminodipropionitrile (IDPN).

In vivo and in vitro release of cyanide from neurotoxic aminonitriles.

Cyanide release from neurotoxic aminonitriles was measured following in vitro incubation with both microsomes and liver slices. Investigation of cyanide released as urinary thiocyanate following ip aminonitrile administration to rats was also measured. The yield of cyanide in the in vivo study, as measured by the mole percent of administered dose, was greatest from dimethylaminonitrile (DMAA), followed by trimethylaminopropionitrile (TMAPN), dimethylaminopropionitrile (DMAPN), 3,3'-iminodipropionitrile (IDPN), dimethylaminobutyronitrile (DMABN), and monomethylaminopropionitrile (MMAPN). Urinary excretion of thiocyanate accounted for 48.9% of the administered DMAA, 11.6% of TMAPN, 8.0% of DMAPN, 6.8% of IDPN, 3.1% of DMABN, and 1.8% of MMAPN. Incubation of aminonitriles and related compounds with microsomes or liver slices from rats yielded measurable quantities of cyanide from all the compounds tested except for DMABN, TMABN, and succiononitrile. Quantitative evaluation of the yield of formaldehyde by demethylation following microsomal incubation was also determined. The signs of acute toxicity in rats after ip administration of KCN were similar only to those in rats administered DMAA.

Development of topical BAPN delivery system for acute spinal cord injury in dogs.

Topical sustained release of various medications by a subdurally implantable device at the site of spinal cord injury is considered advantageous in the treatment of early symptoms of tissue damage. A typical case is the interference with collagenous scar by beta-aminopropionitrile, inhibiting collagen polymerization. Four materials, silicone, polyethylene, polytetrafluoroethylene (PTFE), and polyacrylonitrile-based hydrogel were evaluated for biocompatibility in subcutaneous implantations. The hydrogel, the least reactive, was then compared with silicone sheets as subcural implants. The histology favored the hydrogel as the most inert material, which was then used for the construction of soft, pliable pouches, releasing the drug through the hydrated wall at a rate controlled by an osmotic pump.

Diffusion characteristics of beta-aminopropionitrile in peripheral nerve.

beta-Aminopropionitrile (beta-APN), a lathyrogen, alters the physical characteristics of fibrous scar tissue and as such may have potential clinical use in treatment of injured spinal cord and peripheral nerve by reducing the physical barrier to axon regeneration. For beta-APN to exert its lathyrogenic effect, it must permeate the injury site and gain access to the developing collagenous scar. To investigate the diffusion characteristics, beta-[14C]APN solution was applied as an immersion bath to rat sciatic nerve using both in vivo and in vitro preparations for intervals of 15 to 90 min. The four experimental groups studied were (a) intact nerve, (b) hemisected nerve, (c) nerve with epineurium removed, and (d) nerve with both epineurium and perineurium removed. The isotope labeling index determined by autoradiography and scintillation counting indicated the perineurium as the primary barrier to significant diffusion of beta-APN in normal nerve. When perineurium was incised or removed, beta-APN entered the endoneurial matrix. beta-APN concentration in the epineurium and perineurium increased with increasing bathing time in vitro; but it decreased markedly after 15 min of in vivo bathing. These findings indicate that topical application of beta-APN to injured peripheral nerve would be a successful method of exposing fibrogenic intraneural tissue to the inhibitory effect on lysyl oxidase enzymes. Continuous application, however, will be necessary because of the rapid beta-APN removal documented in the vivo preparation.