Therapeutic strategies to target connective tissue growth factor in fibrotic lung diseases.

The treatment of interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF), remains challenging as current available antifibrotic agents are not effective in halting disease progression. Connective tissue growth factor (CTGF), also known as cellular communication factor 2 (CCN2), is a member of the CCN family of proteins that regulates cell signaling through cell surface receptors such as integrins, the activity of cytokines/growth factors, and the turnover of extracellular matrix (ECM) proteins. Accumulating evidence indicates that CTGF plays a crucial role in promoting lung fibrosis through multiple processes, including inducing transdifferentiation of fibroblasts to myofibroblasts, epithelial-mesenchymal transition (EMT), and cooperating with other fibrotic mediators such as TGF-ß. Increased expression of CTGF has been observed in fibrotic lungs and inhibiting CTGF signaling has been shown to suppress lung fibrosis in several animal models. Thus, the CTGF signaling pathway is emerging as a potential therapeutic target in IPF and other pulmonary fibrotic conditions. This review provides a comprehensive overview of the current evidence on the pathogenic role of CTGF in pulmonary fibrosis and discusses the current therapeutic agents targeting CTGF using a systematic review approach.

Therapeutic strategies targeting pro-fibrotic macrophages in interstitial lung disease.

Idiopathic pulmonary fibrosis (IPF) is the representative phenotype of interstitial lung disease where severe scarring develops in the lung interstitium. Although antifibrotic treatments are available and have been shown to slow the progression of IPF, improved therapeutic options are still needed. Recent data indicate that macrophages play essential pro-fibrotic roles in the pathogenesis of pulmonary fibrosis. Historically, macrophages have been classified into two functional subtypes, "M1″ and "M2," and it is well described that "M2″ or "alternatively activated" macrophages contribute to fibrosis via the production of fibrotic mediators, such as TGF-ß, CTGF, and CCL18. However, highly plastic macrophages may possess distinct functions and phenotypes in the fibrotic lung environment. Thus, M2-like macrophages in vitro and pro-fibrotic macrophages in vivo are not completely identical cell populations. Recent developments in transcriptome analysis, including single-cell RNA sequencing, have attempted to depict more detailed phenotypic characteristics of pro-fibrotic macrophages. This review will outline the role and characterization of pro-fibrotic macrophages in fibrotic lung diseases and discuss the possibility of treating lung fibrosis by preventing or reprogramming the polarity of macrophages. We also utilized a systematic approach to review the literature and identify novel and promising therapeutic agents that follow this treatment strategy.

Monocyte and macrophage derived myofibroblasts: Is it fate? A review of the current evidence.

Since the discovery of the myofibroblast over 50 years ago, much has been learned about its role in wound healing and fibrosis. Its origin, however, remains controversial, with a number of progenitor cells being proposed. Macrophage-myofibroblast transition (MMT) is a recent term coined in 2014 that describes the mechanism through which macrophages, derived from circulating monocytes originating in the bone marrow, transformed into myofibroblasts and contributed to kidney fibrosis. Over the past years, several studies have confirmed the existence of MMT in various systems, suggesting that MMT could potentially occur in all fibrotic conditions and constitute a reasonable therapeutic target to prevent progressive fibrotic disease. In this perspective, we examined recent evidence supporting the notion of MMT in both human disease and experimental models across organ systems. Mechanistic insight from these studies and information from in vitro studies is provided. The findings substantiating plausible MMT showcased the co-expression of macrophage and myofibroblast markers, including CD68 or F4/80 (macrophage) and α-SMA (myofibroblast), in fibroblast-like cells. Furthermore, fate-mapping experiments in murine models exhibiting myeloid-derived myofibroblasts in the tissue further provide direct evidence for MMT. Additionally, we provide some evidence from single cell RNA sequencing experiments confirmed by fluorescent in situ hybridisation studies, showing monocyte/macrophage and myofibroblast markers co-expressed in lung tissue from patients with fibrotic lung disease. In conclusion, MMT is likely a significant contributor to myofibroblast formation in wound healing and fibrotic disease across organ systems. Circulating precursors including monocytes and the molecular mechanisms governing MMT could constitute valid targets and provide insight for the development of novel antifibrotic therapies; however, further understanding of these processes is warranted.

Mouse Models of Lung Fibrosis.

The drug discovery pipeline, from discovery of therapeutic targets through preclinical and clinical development phases, to an approved product by health authorities, is a time-consuming and costly process, where a lead candidates' success at reaching the final stage is rare. Although the time from discovery to final approval has been reduced over the last decade, there is still potential to further optimize and streamline the evaluation process of each candidate as it moves through the different development phases. In this book chapter, we describe our preclinical strategies and overall decision-making process designed to evaluate the tolerability and efficacy of therapeutic candidates suitable for patients diagnosed with fibrotic lung disease. We also describe the benefits of conducting preliminary discovery trials, to aid in the selection of suitable primary and secondary outcomes to be further evaluated and assessed in subsequent internal and external validation studies. We outline all relevant research methodologies and protocols routinely performed by our research group and hope that these strategies and protocols will be a useful guide for biomedical and translational researchers aiming to develop safe and beneficial therapies for patients with fibrotic lung disease.

Activin A and Cell-Surface GRP78 Are Novel Targetable RhoA Activators for Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of kidney failure. RhoA/Rho-associated protein kinase (ROCK) signaling is a recognized mediator of its pathogenesis, largely through mediating the profibrotic response. While RhoA activation is not feasible due to the central role it plays in normal physiology, ROCK inhibition has been found to be effective in attenuating DKD in preclinical models. However, this has not been evaluated in clinical studies as of yet. Alternate means of inhibiting RhoA/ROCK signaling involve the identification of disease-specific activators. This report presents evidence showing the activation of RhoA/ROCK signaling both in vitro in glomerular mesangial cells and in vivo in diabetic kidneys by two recently described novel pathogenic mediators of fibrosis in DKD, activins and cell-surface GRP78. Neither are present in normal kidneys. Activin inhibition with follistatin and neutralization of cell-surface GRP78 using a specific antibody blocked RhoA activation in mesangial cells and in diabetic kidneys. These data identify two novel RhoA/ROCK activators in diabetic kidneys that can be evaluated for their efficacy in inhibiting the progression of DKD.

Cell-specific drug targeting in the lung.

Non-targeted drug delivery systems have several limitations including the decreased bioavailability of the drug, poor stability and rapid clearance in addition to off-target distribution. Cell-specific targeted delivery approaches promise to overcome some of these limitations and enhance therapeutic selectivity. In this review, we aim to discuss cell-specific targeted approachesin the lung at the biochemical and molecular levels. These approaches include;a) directly administered small molecule drugs with intracellular action; b) targeted biologics and synthetic hybrids with extracellular action; c) site activateddrugs; and d) delivery systems.We discuss the pharmaceutical and biochemical parameters that govern the fate of drug molecules at delivery sites while presenting an overview of relevant literature surrounding this area of research and current advancements.

FK506-Binding Protein 13 Expression Is Upregulated in Interstitial Lung Disease and Correlated with Clinical Severity. A Potentially Protective Role.

Pulmonary fibrosis is a progressive lung disease characterized by myofibroblast accumulation and excessive extracellular matrix deposition. We sought to investigate the role of FKBP13 (13-kD FK506-binding protein), an endoplasmic reticulum-resident molecular chaperone, in various forms of pulmonary fibrosis. We first characterized the gene and protein expression of FKBP13 in lung biopsy specimens from 24 patients with idiopathic pulmonary fibrosis and 17 control subjects. FKBP13 expression was found to be elevated in the fibrotic regions of idiopathic pulmonary fibrosis lung tissues and correlated with declining forced vital capacity and dyspnea severity. FKBP13 expression was also increased in lung biopsy specimens of patients with hypersensitivity pneumonitis, rheumatoid arthritis, and sarcoidosis-associated interstitial lung disease. We next evaluated the role of this protein using FKBP13-/- mice in a bleomycin model of pulmonary fibrosis. Animals were assessed for lung function and histopathology at different stages of lung injury including the inflammatory (Day 7), fibrotic (Day 21), and resolution (Day 50) phases. FKBP13-/- mice showed increased infiltration of inflammatory cells and cytokines at Day 7, increased lung elastance and fibrosis at Day 21, and impaired resolution of fibrosis at Day 50. These changes were associated with an increased number of cells that stained positive for TUNEL and cleaved caspase 3 in the FKBP13-/- lungs, indicating a heightened cellular sensitivity to bleomycin. Our findings suggest that FKBP13 is a potential biomarker for severity of interstitial lung diseases and that it has a biologically relevant role in protecting mice against bleomycin-induced injury, inflammation, and fibrosis.

Inhalation: A means to explore and optimize nintedanib's pharmacokinetic/pharmacodynamic relationship.

Oral nintedanib is marketed for the treatment of idiopathic pulmonary fibrosis (IPF). While effective slowing fibrosis progression, as an oral medicine nintedanib is limited. To reduce side effects and maximize efficacy, nintedanib was reformulated as a solution for nebulization and inhaled administration. To predict effectiveness treating IPF, the nintedanib pharmacokinetic/pharmacodynamic relationship was dissected. Pharmacokinetic analysis indicated oral-delivered nintedanib plasma exposure and lung tissue partitioning were not dose-proportional and resulting lung levels were substantially higher than blood. Although initial-oral absorbed nintedanib efficiently partitioned into the lung, only a quickly eliminated fraction appeared available to epithelial lining fluid (ELF). Because IPF disease appears to initiate and progress near the epithelial surface, this observation suggests short duration nintedanib exposure (oral portion efficiently partitioned to ELF) is sufficient for IPF efficacy. To test this hypothesis, exposure duration required for nintedanib activity was explored. In vitro, IPF-cellular matrix (IPF-CM) increased primary normal human fibroblast (nHLF) aggregate size and reduced nHLF cell count. IPF-CM also increased nHLF ACTA2 and COL1A expression. Whether short duration (inhalation pharmacokinetic mimic) or continuous exposure (oral pharmacokinetic mimic), nintedanib (1-100 nM) reversed these effects. In vivo, intubated silica produced a strong pulmonary fibrotic response. Once-daily (QD) 0.021, 0.21 and 2.1 mg/kg intranasal (IN; short duration inhaled exposure) and twice-daily (BID) 30 mg/kg oral (PO; long duration oral exposure) showed that at equivalent-delivered lung exposure, QD short duration inhaled nintedanib (0.21 mg/kg IN vs. 30 mg/kg PO) exhibited equivalent-to-superior activity as BID oral (reduced silica-induced elastance, alpha-smooth muscle actin, interleukin-1 beta (IL-1ß) and soluble collagen). Comparatively, the increased inhaled lung dose (2.1 mg/kg IN vs. 30 mg/kg PO) exhibited increased effect by further reducing silica-induced elastance, IL-1ß and soluble collagen. Neither oral nor inhaled nintedanib reduced silica-induced parenchymal collagen. Both QD inhaled and BID oral nintedanib reduced silica-induced bronchoalveolar lavage fluid macrophage and neutrophil counts with oral achieving significance. In summary, pharmacokinetic elements important for nintedanib activity can be delivered using infrequent, small inhaled doses to achieve oral equivalent-to-superior pulmonary activity.

Protein Misfolding and Endoplasmic Reticulum Stress in Chronic Lung Disease: Will Cell-Specific Targeting Be the Key to the Cure?

Chronic lung disease accounts for a significant global burden with respect to death, disability, and health-care costs. Due to the heterogeneous nature and limited treatment options for these diseases, it is imperative that the cellular and molecular mechanisms underlying the disease pathophysiology are further understood. The lung is a complex organ with a diverse cell population, and each cell type will likely have different roles in disease initiation, progression, and resolution. The effectiveness of a given therapeutic agent may depend on the net effect on each of these cell types. Over the past decade, it has been established that endoplasmic reticulum stress and the unfolded protein response are involved in the development of several chronic lung diseases. These conserved cellular pathways are important for maintaining cellular proteostasis, but their aberrant activation can result in pathology. This review discusses the current understanding of endoplasmic reticulum stress and the unfolded protein response at the cellular level in the development and progression of various chronic lung diseases. We highlight the need for increased understanding of the specific cellular contributions of unfolded protein response activation to these pathologies and suggest that the development of cell-specific targeted therapies is likely required to further decrease disease progression and to promote resolution of chronic lung disease.

Endoplasmic reticulum stress inhibition blunts the development of essential hypertension in the spontaneously hypertensive rat.

Essential hypertension is the leading cause of premature death worldwide. However, hypertension's cause remains uncertain. endoplasmic reticulum (ER) stress has recently been associated with hypertension, but it is unclear whether ER stress causes hypertension. To clarify this question, we examined if ER stress occurs in blood vessels before the development of hypertension and if ER stress inhibition would prevent hypertension development. We used the spontaneously hypertensive rat (SHR) as a model of human essential hypertension and the Wistar-Kyoto (WKY) rat as its normotensive control. Resistance arteries collected from young rats determined that ER stress was present in SHR vessels before the onset of hypertension. To assess the effect of ER stress inhibition on hypertension development, another subset of rats were treated with 4-phenylbutyric acid (4-PBA; 1 g·kg-1·day-1) for 8 wk from 5 wk of age. Blood pressure was measured via radiotelemetry and compared with untreated SHR and WKY rats. Mesenteric resistance arteries were collected and assessed for structural and functional changes associated with hypertension. Systolic and diastolic blood pressures were significantly lower in the 4-PBA-treated SHR groups than in untreated SHRs. Additionally, 4-PBA significantly decreased the media-to-lumen ratio and ER stress marker expression, improved vasodilatory response, and reduced contractile responses in resistance arteries from SHRs. Overall, ER stress inhibition blunted the development of hypertension in the SHR. These data add evidence to the hypothesis that a component of hypertension in the SHR is caused by ER stress. NEW & NOTEWORTHY In this study, 4-phenylbutyric acid's (4-PBA's) molecular chaperone capability was used to inhibit endoplasmic reticulum (ER) stress in the small arteries of young spontaneously hypertensive rats (SHRs) and reduce their hypertension. These effects are likely mediated through 4-PBA's effects to reduce resistant artery contractility and increase nitric oxide-mediated endothelial vasodilation through a process preventing endothelial dysfunction. Overall, ER stress inhibition blunted the development of hypertension in this young SHR model. This suggests that a component of the increase in blood pressure found in SHRs is due to ER stress. However, it is important to note that inhibition of ER stress was not able to fully restore the blood pressure to normal, suggesting that a component of hypertension may not be due to ER stress. This study points to the inhibition of ER stress as an important new physiological pathway to lower blood pressure, where other known approaches may not achieve blood pressure-lowering targets.

Alterations to the middle cerebral artery of the hypertensive-arthritic rat model potentiates intracerebral hemorrhage.

AIMS: We have recently created an age-dependent hypertensive-mono-arthritic animal model from the stroke-resistant spontaneously hypertensive rat to model populations with autoimmune disease who are hypertensive and are prone to stroke. The model exhibits signs of hemorrhagic stroke (HS) subsequent to chronic inflammation and hypertension. HS is also associated with the inability of middle cerebral arteries to undergo pressure dependent constriction (PDC). We investigated alterations in the cerebrovasculature of our hypertensive mono-arthritic animals that develop stroke. MAIN METHODS: Animals were fed either a high salt diet (HSD) (4% NaCl) or Purina chow (0.58% NaCl) from weaning. Complete Freund's Adjuvant (CFA) was injected into the left hind paw at 21-28 weeks; controls received saline and histological and functional studies were performed. RESULTS: Brain damage was more prominent with the high salt, with inflammation exacerbating the damage. High salt alone significantly decreased middle cerebral artery's (MCA's) ability to undergo PDC. Inflammation significantly decreased the ability of cerebrovasculature to respond to pressure step in the regular salt diet. The responses to vasoactive peptides were also significantly attenuated in both inflamed groups regardless of diet. CONCLUSION: Induction of chronic systemic inflammation increases brain damage, and affect the MCA's vasogenic function, decreasing its ability to respond to intraluminal pressure. HSD further exacerbates organ damage associated with chronic inflammation, further compromising cerebrovascular function, and likely increasing the incidence of intracerebral hemorrhage and injury.