Primary Human Lung Alveolus-on-a-chip Model of Intravascular Thrombosis for Assessment of Therapeutics.

Pulmonary thrombosis is a significant cause of patient mortality; however, there are no effective in vitro models of thrombi formation in human lung microvessels that could also assess therapeutics and toxicology of antithrombotic drugs. Here, we show that a microfluidic lung alveolus-on-a-chip lined by human primary alveolar epithelium interfaced with endothelium and cultured under flowing whole blood can be used to perform quantitative analysis of organ-level contributions to inflammation-induced thrombosis. This microfluidic chip recapitulates in vivo responses, including platelet-endothelial dynamics and revealed that lipopolysaccharide (LPS) endotoxin indirectly stimulates intravascular thrombosis by activating the alveolar epithelium, rather than acting directly on endothelium. This model is also used to analyze inhibition of endothelial activation and thrombosis due to a protease activated receptor-1 (PAR-1) antagonist, demonstrating its ability to dissect complex responses and identify antithrombotic therapeutics. Thus, this methodology offers a new approach to study human pathophysiology of pulmonary thrombosis and advance drug development.

Assessment of inhibited alveolar-capillary membrane structural development and function in bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of extreme prematurity and is defined clinically by dependence on supplemental oxygen due to impaired gas exchange. Optimal gas exchange is dependent on the development of a sufficient surface area for diffusion. In the mammalian lung, rapid acquisition of distal lung surface area is accomplished in neonatal and early adult life by means of vascularization and secondary septation of distal lung airspaces. Extreme preterm birth interrupts secondary septation and pulmonary capillary development and ultimately reduces the efficiency of the alveolar-capillary membrane. Although pulmonary health in BPD infants rapidly improves over the first few years, persistent alveolar-capillary membrane dysfunction continues into adolescence and adulthood. Preventative therapies have been largely ineffective, and therapies aimed at promoting normal development of the air-blood barrier in infants with established BPD remain largely unexplored. The purpose of this review will be: (1) to summarize the histological evidence of aberrant alveolar-capillary membrane development associated with extreme preterm birth and BPD, (2) to review the clinical evidence assessing the long-term impact of BPD on alveolar-capillary membrane function, and (3) to discuss the need to develop and incorporate direct measurements of functional gas exchange into clinically relevant animal models of inhibited alveolar development.

Noninvasive assessment of alveolar microvascular recruitment in conscious non-sedated rats.

Recruitment of alveolar microvascular reserves, assessed from the relationship between pulmonary diffusing capacity (DLCO) and perfusion (Q˙c), is critical to the maintenance of arterial blood oxygenation. Leptin-resistant ZDF fatty diabetic (fa/fa) rats exhibit restricted cardiopulmonary physiology under anesthesia. To assess alveolar microvascular function in conscious, non-sedated, non-instrumented, and minimally restrained animals, we adapted a rebreathing technique to study fa/fa and control non-diabetic (+/+) rats (4-5 and 7-11mo old) at rest and during mild spontaneous activity. Measurements included O2 uptake, lung volume, Q˙c, DLCO, membrane diffusing capacity (DMCO), capillary blood volume (Vc) and septal tissue-blood volume. In older fa/fa than +/+ animals, DLCO and DMCO at a given Q˙c were lower; Vc was reduced in proportion to Q˙c. Results demonstrate the consequences of alveolar microangiopathy in the metabolic syndrome: lung volume restriction, reduced Q˙c, and elevated membrane resistance to diffusion. At a given Q˙c, DLCO is lower in rats and guinea pigs than dogs or humans, consistent with limited alveolar microvascular reserves in small animals.

Assessment of alterations in barrier functionality and induction of proinflammatory and cytotoxic effects after sulfur mustard exposure of an in vitro coculture model of the human alveolo-capillary barrier.

Acute lung injury after sulfur mustard (SM) inhalation is characterized by massive, localized hemorrhage and alveolar edema, which implies severe disruption of the vascular and distal airway barrier. In this study, we tested a recently established in vitro coculture model of the alveolo-capillary barrier for its applicability to investigate acute toxic effects of SM at the human respiratory unit. The epithelial compartment of cocultures was exposed to varying concentrations of SM (0-1000 microM; t = 30 min). Following exposure, functional and structural barrier integrity of cocultures was monitored over a period of 24 h. A 50% reduction of transbilayer electrical resistance (TER) within 12-24 h after exposure to 300 microM SM and within 8 h after 1000 microM SM revealed a time- and concentration-dependent impairment of barrier functionality, which was associated with structural loss of both cell layers. Subsequent quantification of interleukin (IL)-6 and IL-8 in cell culture supernatants of exposed cocultures showed enhanced liberation of proinflammatory markers. Highest mediator levels were detected after 300 microM SM, with pronounced stimulation in the endothelial compartment. SM-related cytotoxicity was determined by assessing adenylate kinase (AK) release and by quantifying the fraction of DNA-fragmented nuclei using terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) and nuclear Hoechst staining. Both methods exposed a concentration-dependent increase of SM-mediated cytotoxic effects with high effects on endothelial cells. We conclude that the described in vitro model reflects important characteristics of SM-mediated acute lung injury in vivo and thus can be used to explore involved pathophysiological pathways.