Targeting Longevity Gene SLC13A5: A Novel Approach to Prevent Age-Related Bone Fragility and Osteoporosis.

Reduced expression of the plasma membrane citrate transporter SLC13A5, also known as INDY, has been linked to increased longevity and mitigated age-related cardiovascular and metabolic diseases. Citrate, a vital component of the tricarboxylic acid cycle, constitutes 1-5% of bone weight, binding to mineral apatite surfaces. Our previous research highlighted osteoblasts' specialized metabolic pathway facilitated by SLC13A5 regulating citrate uptake, production, and deposition within bones. Disrupting this pathway impairs bone mineralization in young mice. New Mendelian randomization analysis using UK Biobank data indicated that SNPs linked to reduced SLC13A5 function lowered osteoporosis risk. Comparative studies of young (10 weeks) and middle-aged (52 weeks) osteocalcin-cre-driven osteoblast-specific Slc13a5 knockout mice (Slc13a5cKO) showed a sexual dimorphism: while middle-aged females exhibited improved elasticity, middle-aged males demonstrated enhanced bone strength due to reduced SLC13A5 function. These findings suggest reduced SLC13A5 function could attenuate age-related bone fragility, advocating for SLC13A5 inhibition as a potential osteoporosis treatment.

The citrate transporter SLC13A5 as a therapeutic target for kidney disease: evidence from Mendelian randomization to inform drug development.

BACKGROUND: Solute carrier family 13 member 5 (SLC13A5) is a Na+-coupled citrate co-transporter that mediates entry of extracellular citrate into the cytosol. SLC13A5 inhibition has been proposed as a target for reducing progression of kidney disease. The aim of this study was to leverage the Mendelian randomization paradigm to gain insight into the effects of SLC13A5 inhibition in humans, towards prioritizing and informing clinical development efforts. METHODS: The primary Mendelian randomization analyses investigated the effect of SLC13A5 inhibition on measures of kidney function, including creatinine and cystatin C-based measures of estimated glomerular filtration rate (creatinine-eGFR and cystatin C-eGFR), blood urea nitrogen (BUN), urine albumin-creatinine ratio (uACR), and risk of chronic kidney disease and microalbuminuria. Secondary analyses included a paired plasma and urine metabolome-wide association study, investigation of secondary traits related to SLC13A5 biology, a phenome-wide association study (PheWAS), and a proteome-wide association study. All analyses were compared to the effect of genetically predicted plasma citrate levels using variants selected from across the genome, and statistical sensitivity analyses robust to the inclusion of pleiotropic variants were also performed. Data were obtained from large-scale genetic consortia and biobanks, with sample sizes ranging from 5023 to 1,320,016 individuals. RESULTS: We found evidence of associations between genetically proxied SLC13A5 inhibition and higher creatinine-eGFR (p = 0.002), cystatin C-eGFR (p = 0.005), and lower BUN (p = 3 × 10-4). Statistical sensitivity analyses robust to the inclusion of pleiotropic variants suggested that these effects may be a consequence of higher plasma citrate levels. There was no strong evidence of associations of genetically proxied SLC13A5 inhibition with uACR or risk of CKD or microalbuminuria. Secondary analyses identified evidence of associations with higher plasma calcium levels (p = 6 × 10-13) and lower fasting glucose (p = 0.02). PheWAS did not identify any safety concerns. CONCLUSIONS: This Mendelian randomization analysis provides human-centric insight to guide clinical development of an SLC13A5 inhibitor. We identify plasma calcium and citrate as biologically plausible biomarkers of target engagement, and plasma citrate as a potential biomarker of mechanism of action. Our human genetic evidence corroborates evidence from various animal models to support effects of SLC13A5 inhibition on improving kidney function.

The dual role of citrate in cancer.

Citrate is a key metabolite of the Krebs cycle that can also be exported in the cytosol, where it performs several functions. In normal cells, citrate sustains protein acetylation, lipid synthesis, gluconeogenesis, insulin secretion, bone tissues formation, spermatozoid mobility, and immune response. Dysregulation of citrate metabolism is implicated in several pathologies, including cancer. Here we discuss how cancer cells use citrate to sustain their proliferation, survival, and metastatic progression. Also, we propose two paradoxically opposite strategies to reduce tumour growth by targeting citrate metabolism in preclinical models. In the first strategy, we propose to administer in the tumor microenvironment a high amount of citrate, which can then act as a glycolysis inhibitor and apoptosis inducer, whereas the other strategy targets citrate transporters to starve cancer cells from citrate. These strategies, effective in several preclinical in vitro and in vivo cancer models, could be exploited in clinics, particularly to increase sensibility to current anti-cancer agents.

A Novel and Cross-Species Active Mammalian INDY (NaCT) Inhibitor Ameliorates Hepatic Steatosis in Mice with Diet-Induced Obesity.

Mammalian INDY (mINDY, NaCT, gene symbol SLC13A5) is a potential target for the treatment of metabolically associated fatty liver disease (MAFLD). This study evaluated the effects of a selective, cross-species active, non-competitive, non-substrate-like inhibitor of NaCT. First, the small molecule inhibitor ETG-5773 was evaluated for citrate and succinate uptake and fatty acid synthesis in cell lines expressing both human NaCT and mouse Nact. Once its suitability was established, the inhibitor was evaluated in a diet-induced obesity (DIO) mouse model. DIO mice treated with 15 mg/kg compound ETG-5773 twice daily for 28 days had reduced body weight, fasting blood glucose, and insulin, and improved glucose tolerance. Liver triglycerides were significantly reduced, and body composition was improved by reducing fat mass, supported by a significant reduction in the expression of genes for lipogenesis such as SREBF1 and SCD1. Most of these effects were also evident after a seven-day treatment with the same dose. Further mechanistic investigation in the seven-day study showed increased plasma ß-hydroxybutyrate and activated hepatic adenosine monophosphate-activated protein kinase (AMPK), reflecting findings from Indy (-/-) knockout mice. These results suggest that the inhibitor ETG-5773 blocked citrate uptake mediated by mouse and human NaCT to reduce liver steatosis and body fat and improve glucose regulation, proving the concept of NaCT inhibition as a future liver treatment for MAFLD.

Extracellular citrate and metabolic adaptations of cancer cells.

It is well established that cancer cells acquire energy via the Warburg effect and oxidative phosphorylation. Citrate is considered to play a crucial role in cancer metabolism by virtue of its production in the reverse Krebs cycle from glutamine. Here, we review the evidence that extracellular citrate is one of the key metabolites of the metabolic pathways present in cancer cells. We review the different mechanisms by which pathways involved in keeping redox balance respond to the need of intracellular citrate synthesis under different extracellular metabolic conditions. In this context, we further discuss the hypothesis that extracellular citrate plays a role in switching between oxidative phosphorylation and the Warburg effect while citrate uptake enhances metastatic activities and therapy resistance. We also present the possibility that organs rich in citrate such as the liver, brain and bones might form a perfect niche for the secondary tumour growth and improve survival of colonising cancer cells. Consistently, metabolic support provided by cancer-associated and senescent cells is also discussed. Finally, we highlight evidence on the role of citrate on immune cells and its potential to modulate the biological functions of pro- and anti-tumour immune cells in the tumour microenvironment. Collectively, we review intriguing evidence supporting the potential role of extracellular citrate in the regulation of the overall cancer metabolism and metastatic activity.

The longevity gene INDY (I'm Not Dead Yet) in metabolic control: Potential as pharmacological target.

The regulation of metabolic processes by the Indy (I'm Not Dead Yet) (SLC13A5/NaCT) gene was revealed through studies in Drosophila melanogaster and Caenorhabditis elegans. Reducing the expression of Indy in these species extended their life span by a mechanism resembling caloric restriction, without reducing food intake. In D. melanogaster, mutating the Indy gene reduced body fat content, insulin-like proteins and reactive oxygen species production. Subsequent studies indicated that Indy encodes a citrate transporter located on the cell plasma membrane. The transporter is highly expressed in the mammalian liver. We generated a mammalian knock out model deleting the mammalian homolog mIndy (SLC13A5). The knock out animals were protected from HFD induced obesity, fatty liver and insulin resistance. Moreover, we have shown that inducible and liver selective knock down of mIndy protects against the development of fatty liver and insulin resistance and that obese humans with type 2 diabetes and non-alcoholic fatty liver disease have increased levels of mIndy. Therefore, the transporter mINDY (NaCT) has been proposed to be an 'ideal target for the treatment of metabolic disease'. A small molecule inhibitor of the mINDY transporter has been generated, normalizing glucose levels and reducing fatty liver in a model of diet induced obese mice. Taken together, studies from lower organisms, mammals and humans suggest that mINDY (NaCT) is an attractive target for the treatment of metabolic disease.

α5ß1-Integrin inhibitor (CLT-28643) effective in rabbit trabeculectomy model.

PURPOSE: Glaucoma filtration surgery (GFS) fails due to fibrosis. The α5ß1-integrin plays a pivotal role in fibrosis, angiogenesis and inflammation. This is the first experiment evaluating the prevention of fibrosis after GFS by a specific small molecule α5ß1-integrin inhibitor (CLT-28643). METHODS: Twenty-four rabbits received trabeculectomy on their right eyes. The rabbits were randomized into three groups of eight eyes each. CLT-28643 was given as a single subconjunctival injection intraoperatively to two of the right eye groups followed by postoperative vehicle eye drops (CLT+ group) or CLT-28643 eye drops 4 times daily (CLT++ group). A third group received mitomycin-C (MMC) intraoperatively (sponge application, 0.04%, 2 min) followed by vehicle eye drops postoperatively. The control-surgery group consisted of 12 left eyes having trabeculectomy with no adjunctive therapy. The remaining 12 left eyes formed the untreated group. Clinical assessment included intraocular pressure (IOP) measurement, slit-lamp examination (including bleb survival and morphology) and bleb photography. The rabbits were killed after four weeks for histology. RESULTS: Both CLT-28643-treated groups showed significantly prolonged bleb survival, and better bleb score compared to the control-surgery group. At end of the study, most functioning blebs were found in the MMC group (MMC group 75%; CLT+ group 12.5%, CLT++ group 25%; CLT+ group 12.5%, control-surgery group 0%). CLT-28643 was non-toxic and well tolerated. CONCLUSIONS: This rabbit GFS study indicates that inhibition of α5ß1-integrin by the novel α5ß1-integrin antagonist CLT-28643 significantly improved the outcome. The effect of a single intro-operative application of CLT-28643 seems to be inferior to 0.04% MMC.

Subchoroidal Release of VEGF and bFGF Produces Choroidal Neovascularization in Rabbit.

PURPOSE: Intravitreal vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) produced florid retinal neovascularization and hemorrhage in the rabbit. This study seeks to determine whether sustained subchoroidal release of both VEGF and bFGF can induce robust choroidal neovascularization (CNV) in the rabbit. METHODS: Subchoroidal implantation through the sclera of polymeric pellets containing both 15 µg VEGF and 15 µg bFGF was performed on adult pigmented male Dutch belted rabbits (N = 6) and NZW albinos (N = 8). As negative controls, blank pellets with no growth factors were implanted in both Dutch belted rabbits (N = 6) and NZW albino rabbits (N = 4). Development of CNV was documented weekly over a 4-week period with indirect ophthalmoscopy, color fundus photography, and fluorescein angiography. Eyes were enucleated and prepared for histologic and immunohistochemical analyses at the end of the study. Amounts of VEGF and bFGF that were released in vitro from the pellets were measured by ELISA. RESULTS: In all eyes with subchoroidal implants containing both VEGF and bFGF, strong fluorescein leakage was observed at 2, 3, and 4 weeks (P < 0.005); no leakage was seen initially in week 1. Negative control groups with blank implants showed no fluorescein leakage throughout the 4-week study period. Histologic analysis confirmed the presence of experimental CNV. New subretinal blood vessel growth occurred in all eyes with VEGF/bFGF implants. Negative control eyes with blank implants showed no vascular changes. In vitro sustained release of both VEGF and bFGF was confirmed by ELISA. CONCLUSION: Sustained subchoroidal release of both VEGF and bFGF produced experimental CNV rapidly in the rabbit. Understanding how these growth factors induce CNV may suggest novel therapeutic strategies in the large rabbit eye.

Integrin α5ß1 Inhibition by CLT-28643 Reduces Postoperative Wound Healing in a Mouse Model of Glaucoma Filtration Surgery.

Purpose: To evaluate the therapeutic potential of the small molecule integrin α5ß1 inhibitor, CLT-28643, to improve the filtering surgery outcome in a mouse model. Different dose regimens and administration routes of the inhibitor were compared with mitomycin C (MMC), the gold standard in clin ical practice. Methods: The efficacy of CLT-28643 on surgical outcome was studied in a mouse model for filtering surgery (n = 40 eyes from 20 mice per group). Single and repeated subconjunctival (SCJ) injections (1 or 2 µg) and topical eye drops (10 µg) of the integrin inhibitor were compared with 2-minute administration of MMC 0.02%. Bleb size, survival, and signs of toxicity were examined until 28 days after surgery. Immunohistochemical analysis of angiogenesis, inflammation, collagen deposition, and integrin α5ß1 expression were performed on postoperative days 3, 8, 14, and 28. A masked observer performed all the assessments. Results: Immunostaining showed that integrin α5ß1 was highly expressed in the bleb at early time-points after surgery and that CLT-28643 inhibited this upregulation. Efficacy was shown to be dose-dependent for the integrin inhibitor CLT-28643 for bleb area and survival, and the wound healing process. While 2-µg single injection of CLT-28643 improved bleb characteristics in a similar way as 10-µg administered by eye drops and MMC, repeated injections of 2 µg showed superior efficacy compared to MMC, with no corneal toxicity. Conclusions: Administration of the integrin α5ß1 inhibitor CLT-28643 has therapeutic potential as an adjunct to glaucoma surgery, possibly with a superior efficacy and tolerability compared with MMC when used at the optimal dose.

Inflammation-dependent alpha 5 beta 1 (very late antigen-5) expression on leukocytes reveals a functional role for this integrin in acute peritonitis.

The potential role of alpha 5 beta 1 (VLA-5) in leukocyte trafficking in zymosan-induced acute peritonitis was determined. In naïve mice, approximately 98% of Gr1(high) cells (PMN) in bone marrow and circulation were alpha 5 beta 1-negative; these profiles were modestly affected by peritoneal injection of zymosan. In contrast, approximately 30% of Gr1(high) cells recruited by zymosan (24 h) to the peritoneal cavity expressed alpha 5 beta 1. With respect to F4/80(+) cells, approximately 60% of bone marrow and peripheral blood populations expressed alpha 5 beta 1, with approximately 90% positivity in resident cells of noninflamed peritoneum. Analysis of alpha 5 beta 1 expression revealed inflammation-dependent increased expression on Gr1(high) and F4/80(+) cells in bone marrow, blood, and peritoneal cavity. Blockade of alpha 5 beta 1, by an anti-alpha 5 mAb, attenuated zymosan-induced 24 h recruitment of Gr1(high) and F4/80(+) cells. At least one underlying mechanism of this action was reduction of cell adhesion and transmigration across microvascular vessels, as revealed by intravital microscopy. Confocal analyses indicated that deposition of fibronectin, the principal ligand for alpha 5 beta 1, was up-regulated significantly on and around the inflamed mesenteric microvasculature. These data suggest that the effects of alpha 5-blockade may be a result of inhibition of alpha 5 beta 1-dependent leukocyte adhesion to and migration along the fibronectin matrix. This is the first report that identifies a functional role for alpha 5 beta 1 in leukocyte trafficking during acute inflammation.

Assessment of the integrin alpha5beta1 antagonist JSM6427 in proliferative vitreoretinopathy using in vitro assays and a rabbit model of retinal detachment.

PURPOSE: To explore the role of integrin alpha5beta1 in proliferative vitreoretinopathy (PVR) pathogenesis by evaluating the expression alpha5beta1 on ARPE-19 cells and patient proliferative membranes, quantifying the inhibitory effects of JSM6427 (a small molecule alpha5beta1 inhibitor) on ARPE-19 cell adhesion and migration, and assessing the therapeutic potential of JSM6427 in a rabbit retinal detachment model. METHODS: Expression of alpha5beta1 was evaluated on activated ARPE-19 cells by flow cytometry and on PVR membranes by immunohistochemistry. ARPE-19 cells were used in fibronectin-dependent adhesion and migration assays with various concentrations of JSM6427; IC(50) was calculated. In the rabbit model, eyes were intravitreally injected with vehicle or JSM6427 on day 0 or 1 after retinal detachment; BrdU was administered intravitreally on day 3, and retinal tissues were harvested on day 3 (4 hours later) or 7. Retinal scarring, cellular proliferation, and inflammatory responses were quantified, and retinal morphology was analyzed in retinal sections. RESULTS: Activated ARPE-19 cells and PVR membranes expressed high levels of alpha5beta1; expression was low in control eyes. JSM6427 provided a dose-dependent blockade of ARPE-19 cell adhesion to fibronectin (IC(50), 7.1 +/- 2.5 microM) and inhibition of migration (IC(50), 6.0 +/- 4.5 microM). In the rabbit model, intravitreal injection of JSM6427 provided significant inhibition of proliferation of retinal cells (Müller cells, microglia, and macrophages) on days 3 and 7 after detachment and inhibition of inflammatory response and retinal scarring on day 7 after detachment. CONCLUSIONS: JSM6427 is a promising treatment for PVR, with data suggesting that inhibition of alpha5beta1-fibronectin interactions addresses multiple pathways involving retinal pigment epithelial, glial, and inflammatory cells.

Discovery of orally available integrin alpha5beta1 antagonists.

Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin alpha5beta1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin alpha5beta1 inhibitor scaffolds for potential systemic treatment is described.

Cutting edge: lymphatic vessels, not blood vessels, primarily mediate immune rejections after transplantation.

The purpose of this study was to determine the relative importance of blood vessels (hemangiogenesis) versus lymphatic vessels (lymphangiogenesis) in mediating immunological responses after transplantation. Using the murine model of corneal transplantation, graft survival was compared in differentially prevascularized and avascular recipient beds. Donor corneas (C57BL/6) were transplanted into uninflamed or inflamed avascular, prehemvascularized only or prehemvascularized and prelymphvascularized recipient murine eyes (BALB/C). Selective inhibition of lymphangiogenesis was achieved using antivascular endothelial growth factor receptor 3 Abs and anti-integrin alpha5 small molecules. Grafts placed into only prehemvascularized recipient beds had a similarly good graft survival compared with grafts placed into completely avascular, normal recipients, whereas the pre-existence of lymphatic vessels significantly deteriorated corneal graft survival (p < 0.05). Lymphatic vessels seem to contribute significantly to graft rejection after (corneal) transplantation. That may allow for selective, temporary, perioperative antilymphangiogenic treatment to promote graft survival without affecting blood vessels, even after solid organ transplantation.

SAR of N-phenyl piperidine based oral integrin alpha5beta1 antagonists.

Recently, a new class of selective integrin alpha5beta1inhibitors consisting of a heterocyclic based scaffold was published. Herein the SAR and pharmacokinetic profiles of N-phenyl piperidine derivatives are described.

Preclinical evaluation of the novel small-molecule integrin alpha5beta1 inhibitor JSM6427 in monkey and rabbit models of choroidal neovascularization.

OBJECTIVE: To evaluate the pharmacologic activity and tolerability of JSM6427, a potent and first selective small-molecule inhibitor of integrin alpha5beta1, in monkey and rabbit models of choroidal neovascularization (CNV). METHODS: JSM6427 selectivity for alpha5beta1 was evaluated by in vitro binding assays while the ability of JSM6427 to inhibit CNV was investigated in a laser-induced monkey model and a growth factor-induced rabbit model. Intravitreal injections of JSM6427 (100, 300, or 1000 microg) or vehicle were administered immediately after the CNV induction procedure and at weekly intervals for 4 weeks. Fluorescein angiography was performed weekly. Ocular tolerability was evaluated ophthalmoscopically and histologically in both models; additional assessments in monkeys included electroretinography, biomicroscopy, pathological examination, and analysis of JSM6427 pharmacokinetics. RESULTS: JSM6427 was highly selective for the alpha5beta1-fibronectin interaction. Weekly intravitreal injections of JSM6427 resulted in a statistically significant dose-dependent inhibition of CNV in laser-induced and growth factor-induced models without any ocular JSM6427-related adverse effects. JSM6427 was cleared through the systemic circulation with no evidence of systemic accumulation. CONCLUSIONS: Intravitreal JSM6427 provided dose-dependent inhibition of CNV in monkey and rabbit experimental models. CLINICAL RELEVANCE: JSM6427 may provide a new approach for the treatment of ocular neovascular diseases such as age-related macular degeneration in humans.

Mapping the ligand-binding pocket of integrin alpha5beta1 using a gain-of-function approach.

Integrin alpha5beta1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human integrin alpha5beta1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by integrin alpha5beta1 is poorly understood. In the present study, we demonstrate that zebrafish alpha5beta1 integrins do not interact with human fibronectin or the human alpha5beta1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish alpha5beta1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish alpha5 subunit beta-propeller domain shows that these residues are important for the recognition of the Arg-Gly-Asp (RGD) motif and the synergy sequence [Pro-His-Ser-Arg-Asn (PHSRN)] in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish integrin alpha5 subunit with the corresponding regions of human alpha5 we show that blades 1-4 of the beta-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the beta-propeller domain. We find that the loop connecting blades 2 and 3 of the beta-propeller, the D3-A3 loop, contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human integrin alpha5beta1 supports an important function for D3-A3 loop residues Trp157 and Ala158 in the binding of antagonists. These results will aid the development of reagents that block integrin alpha5beta1 functions in vivo.

Integrin alpha5beta1 mediates attachment, migration, and proliferation in human retinal pigment epithelium: relevance for proliferative retinal disease.

PURPOSE: The aim of this study was to determine the expression and localization of integrin alpha5beta1 in human retinal pigment epithelium (RPE) and its ability to modulate RPE cell attachment, proliferation, migration, and F-actin cytoskeleton distribution. METHODS: Expression and localization of alpha5beta1 were analyzed on human RPE by immunoblot/immunofluorescence. Polarized secretion of fibronectin was measured. RPE attachments to different substrates were determined using cell attachment screening kits. BrdU incorporation and wound-healing assays were used to test hfRPE proliferation and migration. F-actin cytoskeleton was visualized with phalloidin. RESULTS: Integrin alpha5beta1 was detected in native adult and fetal human RPE. The alpha5-subunit is predominantly localized at the apical membrane of hfRPE, whereas the beta1-subunit is uniformly detected at the apical/basolateral membranes. The authors also found that hfRPE cultures secrete significant amounts of fibronectin to the apical bath. JSM6427, a specific integrin alpha5beta1 antagonist, significantly inhibited hfRPE cell attachment to fibronectin, but not laminin, or collagen I or IV. JSM6427 also showed a strong inhibitory effect on bFGF, PDGF-BB, and serum-induced cell migration and proliferation. Furthermore, JSM6427 induced significant disruption of the F-actin cytoskeleton of dividing RPE cells but had no effect on quiescent cells. CONCLUSIONS: The apical localization of alpha5beta1 and the secretion of fibronectin to the apical bath suggest the presence of an autocrine loop that can guide the migration of RPE. The strong inhibitory effects of JSM6427 on human RPE cell attachment, proliferation, and migration is probably mediated by F-actin cytoskeletal disruption in proliferating cells and suggests a potential clinical use of this compound in proliferative retinopathies.

alpha5beta1 Integrin blockade inhibits lymphangiogenesis in airway inflammation.

The integrin alpha5beta1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of alpha5beta1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonis infection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, alpha5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have alpha5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the alpha5beta1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonis infection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth.