Preclinical Core

In vivo rodent models of asthma include the well-established models of allergen-induced allergic lung inflammation using ovalbumin (OVA) or house dust mite (HDM) or its components Dermatophagoides pteronissinus or Dermatophagoides farinae, in sensitization/challenge or challenge protocols. The models, which can be performed in either mice or rats, elicit a predominately eosinophilic, type 2 cytokine dominant allergic lung inflammation accompanied by changes in airway responsiveness to methacholine or other contractile stimuli.  Protocols of longer duration also elicit multiple features of airway remodeling. Drugs/strategies can be tested for their ability to:

1. when administered immediately prior to acute challenge with a contractile agent (e.g., methacholine), inhibit acute bronchoconstriction (i.e., inhibit increases in lung resistance); or
2. when administered prior to or after the allergen treatment protocol, inhibit or reverse the various pathological features of allergic lung inflammation. 

Experimental outcomes include, but are not limited to: multiple indices of lung mechanics including lung resistance and lung compliance, airway cellularity and cytokine abundance, multiple indices of airway remodeling including airway smooth muscle mass, mucous cell metaplasia, and matrix abundance/remodeling. Murine models have an advantage of incorporating genetic strategies (knockout or transgenic mice) whereas rat models are slightly preferable for assessing regulation of remodeling or age-dependent effects.

Related Tissue, Cell-based assays

Cellular assays assessing drug/therapy effects in either human or rodent airway smooth muscle include assays of cellular stiffness/contraction, cytokine production, and matrix production (Ammit et al., 2001; Misior et al., 2008; Misior et al., 2009; Morgan et al., 2014). Assays of human airway epithelia include assays of cytokine production or other induced gene products (e.g., Muc5A), and of cell permeability/barrier function (Park et al., 2008). Additional assays of inflammatory cell (t-cell, mast cell, etc.) function are also possible (Loza et al., 2008). Tissue-based assays include analysis of smooth muscle contractile regulation using precision cut lung slices, or isolated airways using classical organ bath systems, obtained from human or murine lung (Balenga et al., 2014; Morgan et al., 2014).

Related Research References

• Ammit, A. J., A. T. Hastie, L. C. Edsall, R. K. Hoffman, Y. Amrani, V. P. Krymskaya, S. A. Kane, S. P. Peters, R. B. Penn, S. Spiegel and R. A. Panettieri, Jr. (2001) Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. Faseb J. 15(7): 1212-1214.
• Balenga, N. A., W. Jester, M. Jiang, R. A. Panettieri, Jr. and K. M. Druey (2014) Loss of regulator of G protein signaling 5 promotes airway hyperresponsiveness in the absence of allergic inflammation. J Allergy Clin Immunol. 134(2): 451-459.
• Deshpande, D. A., R. M. Pascual, S. W. Wang, D. M. Eckman, E. C. Riemer, C. D. Funk and R. B. Penn (2007) PKC-dependent regulation of the receptor locus dominates functional consequences of cysteinyl leukotriene type 1 receptor activation. FASEB J. 21(10): 2335-2342.
• Forkuo, G. S., H. Kim, V. J. Thanawala, N. Al-Sawalha, D. Valdez, R. Joshi, S. Parra, T. Pera, P. A. Gonnella, B. J. Knoll, J. K. Walker, R. B. Penn and R. A. Bond (2016) PDE4 Inhibitors Attenuate the Asthma Phenotype Produced by beta-adrenoceptor Agonists in PNMT-KO Mice. Am J Respir Cell Mol Biol. 55(2):234-242.
• Liu, T., T. M. Laidlaw, H. R. Katz and J. A. Boyce (2013) Prostaglandin E2 deficiency causes a phenotype of aspirin sensitivity that depends on platelets and cysteinyl leukotrienes. Proc Natl Acad Sci U S A 110(42): 16987-16992.
• Loza, M. J., S. Foster, S. P. Peters and R. B. Penn (2008) Interactive effects of steroids and beta-agonists on accumulation of type 2 T cells. J Allergy Clin.Immunol. 121(3): 750-755.
• Misior, A. M., D. A. Deshpande, M. J. Loza, R. M. Pascual, J. D. Hipp and R. B. Penn (2009) Glucocorticoid- and protein kinase A-dependent transcriptome regulation in airway smooth muscle. Am J Respir Cell Mol Biol. 41(1): 24-39.
• Misior, A. M., H. Yan, R. M. Pascual, D. A. Deshpande, R. A. Panettieri Jr and R. B. Penn (2008) Mitogenic effects of cytokines on smooth muscle are critically dependent on protein kinase A and are unmasked by steroids and cyclooxygenase inhibitors. Mol Pharmacol. 73(2): 566-574.
• Morgan, S. J., D. A. Deshpande, B. C. Tiegs, A. M. Misior, H. Yan, A. V. Hershfeld, T. C. Rich, R. A. Panettieri, S. S. An and R. B. Penn (2014) beta-Agonist-mediated relaxation of airway smooth muscle is protein kinase A-dependent. J Biol Chem. 289(33): 23065-23074.
• Park, J., S. Fang, A. L. Crews, K. W. Lin and K. B. Adler (2008) MARCKS regulation of mucin secretion by airway epithelium in vitro: interaction with chaperones. Am J Respir Cell Mol Biol. 39(1): 68-76.
• Wagner, E. M., J. Jenkins, A. Schmieder, L. Eldridge, Q. Zhang, A. Moldobaeva, H. Zhang, J. S. Allen, X. Yang, W. Mitzner, J. Keupp, S. D. Caruthers, S. A. Wickline and G. M. Lanza (2015) Angiogenesis and airway reactivity in asthmatic Brown Norway rats. Angiogenesis 18(1): 1-11.

In vivo murine models of COPD include two well-established models, the 1) LPS (Pera et al., 2014; Pera et al., 2011) and 2) “smoking” (Kistemaker et al., 2013) mouse models. In the LPS model, animals are subject to  repeated intranasal instillations of LPS (up to 12 weeks). In the smoking mouse model, mice are exposed to cigarette smoke for 4 days. Experimental outcomes for each model include indices of pulmonary inflammation (inflammatory cell counts and cytokines) as well as mucus production, airway fibrosis and emphysema.

Related Tissue, Cell-based Assays

Cellular assays assessing drug/therapy effects of LPS, cigarette smoke, or, cigarette smoke extract (CSE) in human airway smooth muscle include assays of cellular stiffness/contraction, cytokine production, and matrix production that are dysregulated in COPD/emphysema (Pera et al., 2010; Pera et al., 2011; Pera et al., 2012). Assays of human airway epithelia include assays of cytokine production or other induced gene products, and of cell permeability/barrier function (Oldenburger et al., 2014).

Related Research References

• Pera T, Zuidhof AB, Smit M, Menzen MH, Klein T, Flik G, Zaagsma J, Meurs H, Maarsingh H. (2014) Arginase inhibition prevents inflammation and remodeling in a guinea pig model of chronic obstructive pulmonary disease. J Pharmacol Exp Ther. 349(2):229-38.
  
• Pera T, Zuidhof A, Valadas J, Smit M, Schoemaker RG, Gosens R, Maarsingh H, Zaagsma J, Meurs H. (2011)Tiotropium inhibits pulmonary inflammation and remodelling in a guinea pig model of COPD. Eur Respir J. 38(4):789-96.
  
• Kistemaker LE, Bos IS, Hylkema MN, Nawijn MC, Hiemstra PS, Wess J, Meurs H, Kerstjens HA, Gosens R. (2013) Muscarinic receptor subtype-specific effects on cigarette smoke-induced inflammation in mice. Eur Respir J. 42(6):1677-88.
  
• Pera T, Gosens R, Lesterhuis AH, Sami R, van der Toorn M, Zaagsma J, Meurs H. (2010) Cigarette smoke and lipopolysaccharide induce a proliferative airway smooth muscle phenotype. Respir Res. 11:48.
  
• Pera T, Sami R, Zaagsma J, Meurs H. (2011) TAK1 plays a major role in growth factor-induced phenotypic modulation of airway smooth muscle. Am J Physiol Lung Cell Mol Physiol. 301(5):L822-8.
  
• Pera T, Atmaj C, van der Vegt M, Halayko AJ, Zaagsma J, Meurs H. (2012) Role for TAK1 in cigarette smoke-induced proinflammatory signaling and IL-8 release by human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 303(3):L272-8.
  
• Oldenburger A, Poppinga WJ, Kos F, de Bruin HG, Rijks WF, Heijink IH, Timens W, Meurs H, Maarsingh H, Schmidt M. (2014) A-kinase anchoring proteins contribute to loss of E-cadherin and bronchial epithelial barrier by cigarette smoke. Am J Physiol Cell Physiol. 306(6):C585-97.

In vivo murine models of lung fibrosis includes  four different well-established models:

1. The intratracheal bleomycin instillation model. This model is generated by delivering a one-time dose of bleomycin (0.075 units) into posterior oropharyngeal space of anesthetized rodents (Romero et al., 2014). The reversibility of this model makes it an excellent choice for studying both injury and repair mechanisms.
2. The systemic bleomycin delivery model. This model is generated by intravenously administering a single dose of bleomycin (80 mg/kg) or by administering bleomycin via continuous subcutaneous infusion for 7 days using an osmotic minipump (Moore et al., 2013). The reversibility of the single intravenous dose model makes it an excellent choice for studying both injury and repair mechanisms while the subcutaneous model is better for modeling chronic lung disease because fibrosis tends to persist.
3. The silicosis model. This model is generated by instilling a one-time dose of silica particles (20 mg of sterile silica crystal, median diameter 1-5 µm) into the posterior pharynx of anesthetized mice as described previously (Romero et al., 2014).  This model is an excellent model of occupational/environmental-induced chronic fibrotic lung disease.
4. The radiation-induced lung injury. This model is generated by exposing anesthetized mice to a one-time dose of 16 Gy delivered to the thorax while shielding the rest of the body (Romero et al., 2014). This model is an excellent model of radiation-induced lung fibrosis. The disadvantages of this model is the long time between injury and fibrosis (6 months).

Experimental outcomes for each model include, but are not limited to multiple indices of lung mechanics including lung resistance and lung compliance, airway cellularity and cytokine abundance, multiple indices of extracellular matrix abundance and tissue remodeling. Murine models have an advantage of incorporating genetic strategies (knockout or transgenic mice).

Related Tissue, Cell-based Assays

Cellular assays of fibroblast activation employ human and rodent lung fibroblast cell lines to assess effects on cell differentiation, proliferation, and survival and extracellular matrix production. Cellular assays of epithelial cells employ human and rodent alveolar epithelial cells to provide insight into drug effects on growth, survival, proliferation and senescence.

Related Research References

• Romero F, Shah D, Duong M, Penn RB, Fessler MB, Madenspacher J, Stafstrom W, Kavuru M, Lu B, Kallen CB, Walsh K, Summer R. (2014) A Pneumocyte-macrophage paracrine lipid axis drives the lung toward fibrosis. Am J Respir Cell Mol Biol. 53(1):74-86.5
• Moore B, Lawson WE, Oury TD, Sisson TH, Raghavendran K, Hogaboam CM. (2013) Animal models of fibrotic lung disease.  Am J Respir Cell Mol Biol. 49(2):167-79.

In vivo murine models of Acute Lung Injury including systems providing insight into human ARDS pathology. 5 different models are available:

1. The HCl instillation model. This direct lung injury model is generated by instilling a one-time dose of 50 μl of HCl (0.1N) into posterior oropharyngeal space of anesthetized rodents (Shah et al., 2015).
2. The LPS instillation model – This direct lung injury model is generated by instilling a one-time dose of 100 μl of lipopolysaccride (LPS, 100 mcg) into posterior oropharyngeal space of anesthetized rodents (Shah et al., 2015). This model mimics many features of the ARDS, including massive immune cell infiltrations and vascular permeability.
3. The bleomycin model – This direct lung injury model is generated by instilling a one-time dose of 100 μl of bleomycin (bleomycin, 0.05-0.075 units) into posterior oropharyngeal space of anesthetized rodents (Shah et al., 2014). The disadvantages of this model are that fibrotic reactions develops late after instillation and that inflammation tends to predominantly mononuclear at late time points.
4. The oleic acid model – This indirect lung injury model is generated by infusing 70 mg/kg of oleic acid in a bolus form through the left femoral vein of anesthetized rodents (more info ... ). This model has the advantage of injuring the endothelium; however, this model is less clinically relevant.
5. The cecal-ligation and puncture model – This indirect lung injury model is generated by ligating and then puncturing the cecum 3 to 5 times with a needle to induce intra-abdominal sepsis in anesthetized rodents. This is a better model to mimic ARDS caused by systemic sepsis.

Experimental outcomes for each model include, but are not limited to multiple indices of lung mechanics including lung resistance and lung compliance, airway cellularity and cytokine abundance, indices of barrier function including BAL protein concentration, lung wet:dry ratio and junctional protein expression as well as markers of apoptotic and necrotic cell death. Murine models have an advantage of incorporating genetic strategies (knockout or transgenic mice).

Related Tissue, Cell-based Assays

Cellular assays assessing drug/therapy effects on endothelial or epithelial function to LPS, bacteria, drugs (bleomycin), chemicals (HCl) or fatty acids (palmitate) include assays of barrier function, inflammation and cell death using a variety of approaches.

Related Research Publications

• Shah, D., Romero, R., Duong, M., Ying, J., Walsh, K., Summer, R. (2015) C1q Deficiency Promotes Lung Vascular Inflammation and Enhances the Susceptibility of the Lung Endothelium to Injury. J Biol Chem. 290(49):29642-51. 
• Shah, D., Romero, R., Duong, M., Wang, N., Paudyal, B., Suratt, B., Kallen, C.B., Sun, J., Walsh, K., Summer., R. (2015) Obesity-induced adipokine imbalance impairs mouse pulmonary vascular endothelial function and primes the lung for injury. Sci Rep. 5:11362. 
• Shah, D., Romero, F., Stafstrom, W., Duong, M., Summer, R. (2014) Extracellular ATP mediates the late phase of neutrophil recruitment to the lung after LPS induced injury. Am J Respir Cell Mol Biol. 306(2):L152-61.

LPS model of Sepsis entails instilling a one-time dose of 100 μl of lipopolysaccride (LPS, 100 mcg) into the peritoneal cavity of rodents (Doi et al., 2009).  Outcomes include, but are not limited to measurement of serum cytokines, vascular leak, vascular inflammation and organ-specific injury (elevated Cr, hypoxia, liver dysfunction)

Experimental outcomes for each model include, but are not limited to multiple indices of lung mechanics including lung resistance and lung compliance, airway cellularity and cytokine abundance, multiple indices of epithelial and endothelial barrier function. Murine models have an advantage of incorporating genetic strategies (knockout or transgenic mice).

Related Cell-based Assays

Cellular assays assessing drug/therapy effects of LPS, bacterial products, high levels of fatty acids and other agents on mouse and human endothelial cell function employ assays to measure endothelial activation, permeability and cytokine production.

Related Research Publications

• Doi, K., A. Leelahavanichkul, P. S. Yuen and R. A. Star (2009) Animal models of sepsis and sepsis-induced kidney injury. J Clin Invest 119(10): 2868-2878.

Cecal Ligation and Puncture involve ligating and then puncturing the cecum 3 to 5 times with a needle to induce intraabdominal sepsis (Dejager et al., 2011; Pan et al., 2015).  Outcomes include, but are not limited to measurement of serum cytokines, vascular leak, vascular inflammation and organ-specific injury (elevated Cr, hypoxia, liver dysfunction).

Related Cell-based Assays

Cellular assays useful for assessing effects of candidate drugs for treatment of sepsis include blood count, profiling of plasma cytokines, adhesion molecule expression and apoptosis (Weaver et al., 2004; Zhang et al., 2015).

Related Research Publications

• Pan S, Wang N, Bisetto S, Yi B, Sheu SS. (2015) Downregulation of adenine nucleotide translocator 1 exacerbates tumor necrosis factor-α-mediated cardiac inflammatory responses. Am J Physiol Heart Circ Physiol. 308:H39-48.
• Dejager L, Pinheiro I, Dejonckheere E, Libert C. (2011) Cecal ligation and puncture: the gold standard model for polymicrobial sepsis. Trends Microbiol.  4:198-208.
• Zhang J, Yang GM, Zhu Y, Peng XY, Li T, Liu LM (2015) Role of connexin 43 in vascular hyperpermeability and relationship to Rock1-MLC20 pathway in septic rats. Am J Physiol Lung Cell Mol Physiol 309:L1323-L1332.
• Weaver JG, Rouse MS, Steckelberg JM and Badley AD (2004) Improved survival in experimental sepsis with an orally administered inhibitor of apoptosis. FASEB J. 18(11):1185-91.

In vivo models of osteoporosis include mouse or rat estrogen-deficient bone loss generated by bilateral ovariectomy (OVX), which are the most common osteoporotic models (Bouxsein et al., 2005; Masiukiewicz et al., 2000). The OVX mouse model is suitable for the study of pathogenesis of postmenopausal osteoporosis, and various gene knockout mice are available for the experiments. In addition, continuous parathyroid hormone (PTH) administration by infusion pump can cause bone loss and hypercalcemia (Lida-Klein et al., 2005) in mouse or rats. Experimental outcomes include, but are not limited to analyses of bone formation and resorption, and osteoblastogenesis and osteoclastogenesis  (Fig.1).

Related Tissue, Cell-based Assays

Cellular assays assess drug/therapy effects on primary osteoblast mineralization (Wang et al., 2013), osteoclast formation and resorptive activity (Yang et al., 2015) (Fig.2).

Related Research Publications

• Bouxsein, M. L., Myers, K. S., Shultz, K. L., Donahue, L. R., Rosen, C. J., and Beamer, W. G. (2005) Ovariectomy-induced bone loss varies among inbred strains of mice. J Bone and Mineral Res. 20: 1085-1092.
• Masiukiewicz, U. S., Mitnick, M., Grey, A. B., and Insogna, K. L. (2000) Estrogen modulates parathyroid hormone-induced interleukin-6 production in vivo and in vitro. Endocrinology 141: 2526-2531.
• Iida-Klein A, Lu SS, Kapadia R, Burkhart M, Moreno A, Dempster DW, Lindsay R. Short-term continuous infusion of human parathyroid hormone 1-34 fragment is catabolic with decreased trabecular connectivity density accompanied by hypercalcemia in C57BL/J6 mice. J Endocrinol. 186: 549-57.
• Wang B, Yang Y, Liu L, Blair HC, Friedman PA. NHERF1 regulation of PTH-dependent bimodal Pi transport in osteoblasts. Bone 52: 268-77.
• Yang Y, Blair HC, Shapiro IM, Wang B. The Proteasome Inhibitor Carfilzomib Suppresses Parathyroid Hormone-induced Osteoclastogenesis through a RANKL-mediated Signaling Pathway. J Biol Chem. 290: 16918-28.

Models of arthritis include those for osteoarthritis and rheumatoid arthritis.

• Osteorthritis.  In vivo models of osteoarthritis include two separate surgical models, anterior cruciate ligament transection (ACLT; performed in the mouse (Culley et al., 2015)) and destabilization of the medial meniscus (DMM), commonly used to study the onset and progression of posttraumatic osteoarthritis (OA) (Glasson et al., 2007). The DMM model mimics clinical meniscal injury and permits the study of structural and biological changes over the course of the disease. Experimental outcomes for both models include, but are not limited to, analyses of cartilage damage and cartilage hypertrophic markers.
• Rheumatoid arthritis. In vivo models of rheumatoid arthritis include rat adjuvant-induced arthritis (AIA) (Wang et al., 2014), and rat collagen-induced arthritis (CIA) (Godler et al., 2005). AIA is induced by intradermal injection of inactive Bacillus Calmette-Guérin suspended in paraffin oil  into the hind food pat. The hallmarks of AIA are reliable onset of robust polyarticular inflammation and marked bone resorption. CIA is induced by intradermal injection of type II collagen emulsified in incomplete Freund's adjuvant at the base of the tail. CIA is polyarthritis characterized by marked cartilage destruction associated with immune complex deposition on articular surfaces and bone resorption.

Related Tissue, Cell-based Assays

Relevant cellular assays include assessment of chondrogenic differentiation using a chondrogenic cell line (ATDC5 cells)(Fig. 3) or chondrocytes derived from mesenchymal stem cells, and assays of cartilage proteoglycan synthesis. Cell based assays of rheumatoid arthritis include macrophages isolated from peritoneal lavage, synoviocytes prepared from synovial membranes of knee joints, and lymphocytes isolated from spleens for studies of drug efficacy on inflammatory cytokine production and immune response (Wang et al., 2014).

Related Research Publications

• Culley, K. L., Dragomir, C. L., Chang, J., Wondimu, E. B., Coico, J., Plumb, D. A., Otero, M., and Goldring, M. B. (2015) Mouse models of osteoarthritis: surgical model of posttraumatic osteoarthritis induced by destabilization of the medial meniscus. Methods in molecular biology 1226: 143-173.
• Glasson, S. S., Blanchet, T. J., and Morris, E. A. (2007) The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage 15: 1061-1069.
• Godler DE, Stein AN, Bakharevski O, Lindsay MM, Ryan PF (2005) Parathyroid hormone-related peptide expression in rat collagen-induced arthritis. Rheumatology (Oxford) 44: 1122-1131.
• Wang, B., and Chen, M. Z. (2014) Astragaloside IV possesses antiarthritic effect by preventing interleukin 1beta-induced joint inflammation and cartilage damage. Arch Pharm Res. 37: 793-802.