Human Vascular Wall Microfluidic Model for Preclinical Evaluation of Drug-Induced Vascular Injury.

Drug-induced vascular injury (DIVI) in preclinical animal models often leads to candidate compound termination during drug development. DIVI has not been documented in human clinical trials with drugs that cause DIVI in preclinical animals. A robust human preclinical assay for DIVI is needed as an early vascular injury screen. A human vascular wall microfluidic tissue chip was developed with a human umbilical vein endothelial cell (HUVEC)-umbilical artery smooth muscle cell (vascular smooth muscle cell, VSMC) bilayer matured under physiological shear stress. Optimized temporal flow profiles produced HUVEC-VSMC bilayers with quiescent endothelial cell (EC) monolayers, EC tight junctions, and contractile VSMC morphology. Dose-response testing (3-30 µM concentration) was conducted with minoxidil and tadalafil vasodilators. Both drugs have demonstrated preclinical DIVI but lack clinical evidence. The permeability of severely damaged engineered bilayers (30 µM tadalafil) was 4.1 times that of the untreated controls. Immunohistochemical protein assays revealed contrasting perspectives on tadalafil and minoxidil-induced damage. Tadalafil impacted the endothelial monolayer with minor injury to the contractile VSMCs, whereas minoxidil demonstrated minor EC barrier injury but damaged VSMCs and activated ECs in a dose-response manner. This proof-of-concept human vascular wall bilayer model of DIVI is a critical step toward developing a preclinical human screening assay for drug development. Impact statement More than 90% of drug candidates fail during clinical trials due to human efficacy and toxicity concerns. Preclinical studies rely heavily on animal models, although animal toxicity and drug metabolism responses often differ from humans. During the drug development process, perfused in vitro human tissue chips could model the clinical drug response and potential toxicity of candidate compounds. Our long-term objective is to develop a human vascular wall tissue chip to screen for drug-induced vascular injury. Its application could ultimately reduce drug development delays and costs, and improve patient safety.

Vascular Imaging of Matrix Metalloproteinase Activity as an Informative Preclinical Biomarker of Drug-induced Vascular Injury.

Lack of biomarkers specific to and either predictive or diagnostic of drug-induced vascular injury (DIVI) continues to be a major obstacle during drug development. Biomarkers derived from physiologic responses to vessel injury, such as inflammation and vascular remodeling, could make good candidates; however, they characteristically lack specificity for vasculature. We evaluated whether vascular remodeling-associated protease activity, as well as changes to vessel permeability resulting from DIVI, could be visualized ex vivo in affected vessels, thereby allowing for visual monitoring of the pathology to address specificity. We found that visualization of matrix metalloproteinase activation accompanied by increased vascular leakage in the mesentery of rats treated with agents known to induce vascular injury correlated well with incidence and severity of histopathological findings and associated inflammation as well as with circulating levels of tissue inhibitors of metalloproteinase 1 and neutrophil gelatinase-associated lipocalin. The weight of evidence approach reported here shows promise as a composite DIVI preclinical tool by means of complementing noninvasive monitoring of circulating biomarkers of inflammation with direct imaging of affected vasculature and thus lending specificity to its interpretation. These findings are supportive of a potential strategy that relies on translational imaging tools in conjunction with circulating biomarker data for high-specificity monitoring of VI both preclinically and clinically.

The Far Side of Vascular Injury: Nonconventional Vasoconstrictors, DNA-targeting Agents, and Agents Toxic to Vascular Smooth Muscle.

Substances historically thought to cause direct vascular injury in laboratory animals are a heterogeneous group of toxic agents with varied mechanisms of action. Morphologically, the reviewed agents can be broadly categorized into those targeting endothelial cell (ECs) and those targeting smooth muscle cells (SMCs). Anticancer drugs, immunosuppressants, and heavy metals are targeting primarily ECs while allylamine, ß-aminopropionitrile, and mitogen-activated protein kinase kinase inhibitors affect mainly SMCs. It is now recognized that the pathogenicity of some of these agents is often mediated through intermediary events, particularly vasoconstriction. There are clear similarities in the clinical and microscopic findings associated with many of these agents in animals and man, allowing the use of animal models to investigate mechanisms and pathogenesis. The molecular pathogenic mechanisms and comparative morphology in animals and humans will be reviewed.

L-citrulline for protection of endothelial function from ADMA-induced injury in porcine coronary artery.

Endogenous nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine (ADMA) is a cardiovascular risk factor. We tested the hypothesis that L-citrulline may ameliorate the endothelial function altered by ADMA in porcine coronary artery (PCA). Myograph study for vasorelaxation, electrochemical measurement for NO, RT-PCR, and Western blot analysis for expression of eNOS, argininosuccinate synthetase (ASS), and p-eNOS(ser1177) were performed. cGMP was determined by enzyme immunoassay. Superoxide anion (O2.(-)) production was detected by the lucigenin-enhanced chemiluminescence method. Compare with controls (96.03% ± 6.2%), the maximal relaxation induced by bradykinin was significantly attenuated (61.55% ± 4.8%, p<0.01), and significantly restored by L-citrulline (82.67 ± 6.4%, p<0.05) after 24 hours of ADMA exposure. Expression of eNOS, p-eNOS(ser1177), and ASS in PCA significantly increased after L-citrulline incubation. L-citrulline also markedly restored the NO production, and cGMP level which was reduced by ADMA. The increased O2.(-) production by ADMA was also inhibited by L-citrulline. L-citrulline restores the endothelial function in preparations treated with ADMA by preservation of NO production and suppression of O2.(-) generation. Preservation of NO is attributed to the upregulation of eNOS expression along with activation of p-eNOS(ser1177). L-citrulline improves endothelium-dependent vasodilation through NO/ cGMP pathway.

Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part I. Biotherapeutics.

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. Although DIVI in laboratory animal species has been well characterized for vasoactive small molecules, there is little available information regarding DIVI associated with biotherapeutics such as peptides/proteins or antibodies. Because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans and the lack of robust biomarkers of DIVI, preclinical DIVI findings can cause considerable delays in or even halt development of promising new drugs. This review discusses standard terminology, characteristics, and mechanisms of DIVI associated with biotherapeutics. Guidance and points to consider for the toxicologist and pathologist facing preclinical cases of biotherapeutic-related DIVI are outlined, and examples of regulatory feedback for each of the mechanistic types of DIVI are included to provide insight into risk assessment.

Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part 2. Antisense Oligonucleotides.

Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. In recent years, DIVI has been occasionally observed in nonhuman primates given RNA-targeting therapeutics such as antisense oligonucleotide therapies (ASOs) during chronic toxicity studies. While DIVI in laboratory animal species has been well characterized for vasoactive small molecules, and immune-mediated responses against large molecule biotherapeutics have been well described, there is little published information regarding DIVI induced by ASOs to date. Preclinical DIVI findings in monkeys have caused considerable delays in development of promising new ASO therapies, because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans, and the lack of robust biomarkers of DIVI. This review of DIVI discusses clinical and microscopic features of vasculitis in monkeys, their pathogenic mechanisms, and points to consider for the toxicologist and pathologist when confronted with ASO-related DIVI. Relevant examples of regulatory feedback are included to provide insight into risk assessment of ASO therapies.

Aspirin enhances protective effect of fish oil against thrombosis and injury-induced vascular remodelling.

BACKGROUND AND PURPOSE: Although aspirin (acetylsalicylic acid) is commonly used to prevent ischaemic events in patients with coronary artery disease, many patients fail to respond to aspirin treatment. Dietary fish oil (FO), containing ω3 polyunsaturated fatty acids (PUFAs), has anti-inflammatory and cardio-protective properties, such as lowering cholesterol and modulating platelet activity. The objective of the present study was to investigate the potential additional effects of aspirin and FO on platelet activity and vascular response to injury. EXPERIMENTAL APPROACH: Femoral arterial remodelling was induced by wire injury in mice. Platelet aggregation, and photochemical- and ferric chloride-induced carotid artery thrombosis were employed to evaluate platelet function. KEY RESULTS: FO treatment increased membrane ω3 PUFA incorporation, lowered plasma triglyceride and cholesterol levels, and reduced systolic BP in mice. FO or aspirin alone inhibited platelet aggregation; however, when combined, they exhibited synergistic suppression of platelet activity in mice, independent of COX-1 inhibition. FO alone, but not aspirin, attenuated arterial neointimal growth in response to injury. Strikingly, a combination of FO and aspirin synergistically inhibited injury-induced neointimal hyperplasia and reduced perivascular inflammatory reactions. Moreover, co-administration of FO and aspirin decreased the expression of pro-inflammatory cytokines and adhesion molecules in inflammatory cells. Consistently, a pro-resolution lipid mediator-Resolvin E1, was significantly elevated in plasma in FO/aspirin-treated mice. CONCLUSIONS AND IMPLICATIONS: Co-administration of FO and low-dose aspirin may act synergistically to protect against thrombosis and injury-induced vascular remodelling in mice. Our results support further investigation of adjuvant FO supplementation for patients with stable coronary artery disease.

Translation strategy for the qualification of drug-induced vascular injury biomarkers.

Drug-induced vascular injury (DIVI) is a common preclinical toxicity usually characterized by hemorrhage, vascular endothelial and smooth muscle damage, and inflammation. DIVI findings can cause delays or termination of drug candidates due to low safety margins. The situation is complicated by the absence of sensitive, noninvasive biomarkers for monitoring vascular injury and the uncertain relevance to humans. The Safer And Faster Evidence-based Translation (SAFE-T) consortium is a public-private partnership funded within the European Commission's Innovative Medicines Initiative (IMI) aiming to accelerate drug development by qualifying biomarkers for drug-induced organ injuries, including DIVI. The group is using patients with vascular diseases that have key histomorphologic features (endothelial damage, smooth muscle damage, and inflammation) in common with those observed in DIVI, and has selected candidate biomarkers associated with these features. Studied populations include healthy volunteers, patients with spontaneous vasculitides and other vascular disorders. Initial results from studies with healthy volunteers and patients with vasculitides show that a panel of biomarkers can successfully discriminate the population groups. The SAFE-T group plans to seek endorsement from health authorities (European Medicines Agency and Food and Drug Administration) to qualify the biomarkers for use in regulatory decision-making processes.

Nonclinical safety biomarkers of drug-induced vascular injury: current status and blueprint for the future.

Better biomarkers are needed to identify, characterize, and/or monitor drug-induced vascular injury (DIVI) in nonclinical species and patients. The Predictive Safety Testing Consortium (PSTC), a precompetitive collaboration of pharmaceutical companies and the U.S. Food and Drug Administration (FDA), formed the Vascular Injury Working Group (VIWG) to develop and qualify translatable biomarkers of DIVI. The VIWG focused its research on acute DIVI because early detection for clinical and nonclinical safety monitoring is desirable. The VIWG developed a strategy based on the premise that biomarkers of DIVI in rat would be translatable to humans due to the morphologic similarity of vascular injury between species regardless of mechanism. The histomorphologic lexicon for DIVI in rat defines degenerative and adaptive findings of the vascular endothelium and smooth muscles, and characterizes inflammatory components. We describe the mechanisms of these changes and their associations with candidate biomarkers for which advanced analytical method validation was completed. Further development is recommended for circulating microRNAs, endothelial microparticles, and imaging techniques. Recommendations for sample collection and processing, analytical methods, and confirmation of target localization using immunohistochemistry and in situ hybridization are described. The methods described are anticipated to aid in the identification and qualification of translational biomarkers for DIVI.

Quantitative evaluation of drug-induced microvascular constriction in mice kidney using a novel tool for 3D geometrical analysis of ex vivo organ vasculature.

The analysis of organ vasculature, and more specifically organ microvasculature, carries special importance for toxicological sciences, and especially for evaluation of drug-induced vascular toxicity. This field presents a special challenge in nonclinical drug safety assessments since there are currently no reliable microvascular toxicity biomarkers. Therefore, we aimed to systematically investigate the use of microvascular 3D geometrical analysis of corrosion casts for evaluation of drug-induced vascular toxicity, utilizing a novel image investigation tool that allows full 3D-quantified geometrical analysis of the entire vascular tree structure. Vascular casts of kidneys from control and low- and high-dose ephedrine/caffeine-treated mice were scanned by a micro CT, and images were processed and analyzed using the Vasculomics™ platform. All evaluations were performed on the kidney cortex. Treatment resulted in a significant and dose-related reduction in overall microvessel density throughout the kidney cortex. This effect was most pronounced for vessels with diameters between 25 µm and 35 µm, and affected mostly vessels located in the superficial part of the kidney cortex. The use of 3D analysis tools in drug-induced vascular toxicity studies allows for very high resolution and characterization of drug effects on the microvasculature and can be used as a valuable tool in drug safety assessments.

Vascular origin of vildagliptin-induced skin effects in Cynomolgus monkeys: pathomechanistic role of peripheral sympathetic system and neuropeptide Y.

The purpose of this article is to characterize skin lesions in cynomolgus monkeys following vildagliptin (dipeptidyl peptidase-4 inhibitor) treatment. Oral vildagliptin administration caused dose-dependent and reversible blister formation, peeling and flaking skin, erosions, ulcerations, scabs, and sores involving the extremities at ≥5 mg/kg/day and necrosis of the tail and the pinnae at ≥80 mg/kg/day after 3 weeks of treatment. At the affected sites, the media and the endothelium of dermal arterioles showed hypertrophy/hyperplasia. Skin lesion formation was prevented by elevating ambient temperature. Vildagliptin treatment also produced an increase in blood pressure and heart rate likely via increased sympathetic tone. Following treatment with vildagliptin at 80 mg/kg/day, the recovery time after lowering the temperature in the feet of monkeys and inducing cold stress was prolonged. Ex vivo investigations showed that small digital arteries from skin biopsies of vildagliptin-treated monkeys exhibited an increase in neuropeptide Y-induced vasoconstriction. This finding correlated with a specific increase in NPY and in NPY1 receptors observed in the skin of vildagliptin-treated monkeys. Present data provide evidence that skin effects in monkeys are of vascular origin and that the effects on the NPY system in combination with increased peripheral sympathetic tone play an important pathomechanistic role in the pathogenesis of cutaneous toxicity.