High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy.

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy.

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles (OMVs), have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation co-isolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). E. coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

Impact of storage conditions and duration on function of native and cargo-loaded mesenchymal stromal cell extracellular vesicles.

BACKGROUND AIMS: As evidenced by ongoing clinical trials and increased activity in the commercial sector, extracellular vesicle (EV)-based therapies have begun the transition from bench to bedside. As this progression continues, one critical aspect of EV clinical translation is understanding the effects of storage and transport conditions. Several studies have assessed the impact of storage on EV characteristics such as morphology, uptake and component content, but effects of storage duration and temperature on EV functional bioactivity and, especially, loaded cargo are rarely reported. METHODS: The authors assessed EV outcomes following storage at different temperatures (room temperature, 4°C, -20°C, -80°C) for various durations as well as after lyophilization. RESULTS: Mesenchymal stromal cell (MSC) EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4-6 weeks at -20°C and -80°C and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA and long non-coding RNA cargo in MSC EVs. CONCLUSIONS: These findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storing MSC EVs for both basic research and translational purposes.

Co-Targeting ErbB Receptors and the PI3K/AKT Axis in Androgen-Independent Taxane-Sensitive and Taxane-Resistant Human Prostate Cancer Cells.

Using two representative models of androgen-independent prostate cancer (PCa), PC3 and DU145, and their respective paclitaxel- and docetaxel-resistant derivatives, we explored the anti-tumor activity of targeting the ErbB receptors and AKT using small-molecule kinase inhibitors. These cells manifest varying degrees of neuroendocrine differentiation characteristics and differ in their expression of functional PTEN. Although the specific downstream signaling events post the ErbB receptor and AKT co-targeting varied between the PC3- and DU145-lineage cells, synergistic anti-proliferative and enhanced pro-apoptotic responses occurred across the wild-type and the taxane-resistant cells, independent of their basal AKT activation state, their degree of paclitaxel- or docetaxel-resistance, or whether this resistance was mediated by the ATP Binding Cassette transport proteins. Dual targeting also led to enhanced anti-tumor responses in vivo, although there was pharmacodynamic discordance between the PCa cells in culture versus the tumor xenografts in terms of the relative activation and inhibition states of AKT and ERK under basal conditions and upon AKT and/or ErbB targeting. The consistent inhibition, particularly of AKT, occurred both in vitro and in vivo, independent of the underlying PTEN status. Thus, co-targeting AKT with ErbB, and possibly other partners, may be a useful strategy to explore further for potential therapeutic effect in advanced PCa.

Sustained released of bioactive mesenchymal stromal cell-derived extracellular vesicles from 3D-printed gelatin methacrylate hydrogels.

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate the therapeutic potential of MSC EV-laden GelMA bioinks.

HOTAIR-Loaded Mesenchymal Stem/Stromal Cell Extracellular Vesicles Enhance Angiogenesis and Wound Healing.

Chronic wounds remain a substantial source of morbidity worldwide. An emergent approach that may be well-suited to induce the complex, multicellular processes such as angiogenesis that are required for wound repair is the use of extracellular vesicles (EVs). EVs contain a wide variety of proteins and nucleic acids that enable multifactorial signaling. Here, the capability of EVs is leveraged to be engineered via producer cell modification to investigate the therapeutic potential of EVs from mesenchymal stem/stromal cells (MSCs) transfected to overexpress long non-coding RNA HOX transcript antisense RNA (HOTAIR). HOTAIR is previously shown by the authors' group to be critical in mediating angiogenic effects of endothelial cell EVs, and MSCs are chosen as EV producer cells for this study due to their widely reported intrinsic angiogenic properties. The results indicate that MSCs overexpressing HOTAIR (HOTAIR-MSCs) produce EVs with increased HOTAIR content that promote angiogenesis and wound healing in diabetic (db/db) mice. Further, endothelial cells exposed to HOTAIR-MSC EVs exhibit increased HOTAIR content correlated with upregulation of the angiogenic protein vascular endothelial growth factor. Thus, this study supports EV-mediated HOTAIR delivery as a strategy for further exploration toward healing of chronic wounds.

Therapeutic potential of extracellular vesicle-associated long noncoding RNA.

Both extracellular vesicles (EVs) and long noncoding RNAs (lncRNAs) have been increasingly investigated as biomarkers, pathophysiological mediators, and potential therapeutics. While these two entities have often been studied separately, there are increasing reports of EV-associated lncRNA activity in processes such as oncogenesis as well as tissue repair and regeneration. Given the powerful nature and emerging translational impact of other noncoding RNAs such as microRNA (miRNA) and small interfering RNA, lncRNA therapeutics may represent a new frontier. While EVs are natural vehicles that transport and protect lncRNAs physiologically, they can also be engineered for enhanced cargo loading and therapeutic properties. In this review, we will summarize the activity of lncRNAs relevant to both tissue repair and cancer treatment and discuss the role of EVs in enabling the potential of lncRNA therapeutics.

Extracellular miR-146a-5p Induces Cardiac Innate Immune Response and Cardiomyocyte Dysfunction.

Previous studies have demonstrated that transient myocardial ischemia leads to release of cellular nucleic acids such as RNA. Extracellular RNA reportedly plays a pivotal role in myocardial inflammation and ischemic injury in animals. RNA profiling has identified that numerous microRNA (miRNAs), such as ss-miR-146a-5p, are upregulated in plasma following myocardial ischemia, and certain uridine-rich miRNAs exhibit strong proinflammatory effects in immune cells via ssRNA-sensing mechanism. However, the effect of extracellular miRNAs on myocardial inflammation and cardiac cell function remains unknown. In this study, we treated adult mouse cardiomyocytes with miR-146a-5p loaded in extracellular vesicles and observed a dose- and TLR7-dependent production of CXCL-2, IL-6, and TNF-α. In vivo, a single dose of myocardial injection of miR-146a-5p induced both cytokine expression (CXCL2, IL-6, and TNF-α) and innate immune cell activation (CD45+ leukocytes, Ly6Cmid+ monocytes, Ly6G+ neutrophils), which was significantly attenuated in the hearts of TLR7 KO mice. We discovered that conditioned media from miR-146a-treated macrophages stimulated proinflammatory cytokine production in adult cardiomyocytes and significantly inhibited their sarcomere shortening. Finally, using an electric cell impedance-sensing assay, we found that the conditioned media from miR-146a-treated cardiac fibroblasts or cardiomyocytes impaired the barrier function of coronary artery endothelial cells. Taken together, these data demonstrate that extracellular miR-146a-5p activates multiple cardiac cells and induces myocardial inflammation and cardiomyocyte dysfunction via intercellular interaction and innate immune TLR7 nucleic acid sensing.

Homologous Quorum Sensing Regulatory Circuit: A Dual-Input Genetic Controller for Modulating Quorum Sensing-Mediated Protein Expression in E. coli.

We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in three modes: a constitutive mode that enables consistent and high levels of expression; a tightly repressed mode in which there is very little background expression; and an inducible mode in which concentrations of two signals (arabinose and autoinducer-2) determine the net amplification of the gene(s)-of-interest. We demonstrate the utility of the circuit for the controlled expression of human granulocyte macrophage colony stimulating factor in an engineered probiotic E. coli. This dual-input genetic controller is the first homologous AI-2 quorum sensing circuit that has the ability to be operated in three different modes. We believe it has the potential for wide-ranging biotechnological applications due its versatile features.

3D printed HUVECs/MSCs cocultures impact cellular interactions and angiogenesis depending on cell-cell distance.

Vascularization is a crucial process during the growth and development of bone 1, yet it remains one of the main challenges in the reconstruction of large bone defects. The use of in vitro coculture of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) has been one of the most explored options. Both cell types secrete specific growth factors that are mutually beneficial, and studies suggested that cell-cell communication and paracrine secretion could be affected by a number of factors. However, little is known about the effect of cell patterning and the distance between cell populations on their crosstalk. In the present study, we showed that the separation and distance between ECs and MSCs populations affects angiogenesis by modulating cell-cell communication. HUVECs grown farther apart from MSCs (˃400 µm) presented characteristics of an early stage of angiogenesis (migration/proliferation). Results showed an increase in the up-regulation of VEGF, FGF-2, and ITGA3 (integrins) but a smaller fold change in the expression of VE-Cadherin and Ang-1. HUVECs were also still highly proliferative. On the contrary, HUVECs incubated closer (≤200 µm) to MSCs, showed signs of stabilization, mainly an increase in Ang-1 and VE-cadherin expression, as well as tighter monolayers. Conditioned media collected from HUVECs and MSCs grown ≤200 µm apart preferentially promoted tube formation, a later stage of angiogenesis, due in part to a significant increase in Ang-1 paracrine secretion. In addition, in groups in which fibers were printed farther apart (400 µm), cells produced EVs with a significantly increase cargo. Finally, in vivo experiment results showed an increase in blood vessels density and new bone thickness after 12 weeks of implantation in rat cranial defect, further suggesting the higher efficiency of indirect ECs/MSCs contact in prompting the release of paracrine signals that stimulate the angiogenesis of local tissues, and enhanced subsequent bone regeneration.

Cardiac regeneration using human-induced pluripotent stem cell-derived biomaterial-free 3D-bioprinted cardiac patch in vivo.

One of the leading causes of death worldwide is heart failure. Despite advances in the treatment and prevention of heart failure, the number of affected patients continues to increase. We have recently developed 3D-bioprinted biomaterial-free cardiac tissue that has the potential to improve cardiac function. This study aims to evaluate the in vivo regenerative potential of these 3D-bioprinted cardiac patches. The cardiac patches were generated using 3D-bioprinting technology in conjunction with cellular spheroids created from a coculture of human-induced pluripotent stem cell-derived cardiomyocytes, fibroblasts, and endothelial cells. Once printed and cultured, the cardiac patches were implanted into a rat myocardial infarction model (n = 6). A control group (n = 6) without the implantation of cardiac tissue patches was used for comparison. The potential for regeneration was measured 4 weeks after the surgery with histology and echocardiography. 4 weeks after surgery, the survival rates were 100% and 83% in the experimental and the control group, respectively. In the cardiac patch group, the average vessel counts within the infarcted area were higher than those within the control group. The scar area in the cardiac patch group was significantly smaller than that in the control group. (Figure S1) Echocardiography showed a trend of improvement of cardiac function for the experimental group, and this trend correlated with increased patch production of extracellular vesicles. 3D-bioprinted cardiac patches have the potential to improve the regeneration of cardiac tissue and promote angiogenesis in the infarcted tissues and reduce the scar tissue formation.

HER3-Targeted Affibodies with Optimized Formats Reduce Ovarian Cancer Progression in a Mouse Xenograft Model.

Expression of the receptor tyrosine kinase HER3 is negatively correlated with survival in ovarian cancer, and HER3 overexpression is associated with cancer progression and therapeutic resistance. Thus, improvements in HER3-targeted therapy could lead to significant clinical impact for ovarian cancer patients. Previous work from our group established multivalency as a potential strategy to improve the therapeutic efficacy of HER3-targeted ligands, including affibodies. Others have established HER3 affibodies as viable and potentially superior alternatives to monoclonal antibodies for cancer therapy. Here, bivalent HER3 affibodies were engineered for optimized production, specificity, and function as evaluated in an ovarian cancer xenograft model. Enhanced inhibition of HER3-mediated signaling and increased HER3 downregulation associated with multivalency could be achieved with a simplified construct, potentially increasing translational potential. Additionally, functional effects of affibodies due to multivalency were found to be specific to HER3 targeting, suggesting a unique molecular mechanism. Further, HER3 affibodies demonstrated efficacy in ovarian cancer xenograft mouse models, both as single agents and in combination with carboplatin. Overall, these results reinforce the potential of HER3-targeted affibodies for cancer therapy and establish treatment of ovarian cancer as an application where multivalent HER3 ligands may be useful. Further, this work introduces the potential of HER3 affibodies to be utilized as part of clinically relevant combination therapies (e.g., with carboplatin).

Circulating Plasma Extracellular Vesicles from Septic Mice Induce Inflammation via MicroRNA- and TLR7-Dependent Mechanisms.

We have previously reported that a group of host cellular microRNAs (miRNAs; miR-34a-5p, miR-122-5p, miR-145-5p, miR-146a-5p, miR-210-3p) are released into the blood during sepsis, some of which are capable of inducing complement activation, cytokine production, and leukocyte migration. Extracellular vesicles (EVs) have been proposed as vehicles for extracellular miRNA-mediated intercellular communication. However, the biological function of plasma EVs and the associated miRNAs in sepsis are largely unknown. In this study, we tested the hypothesis that plasma EVs in sepsis are proinflammatory and EV-associated miRNAs are responsible for EV-induced cytokine production. Compared with those of sham mice, the plasma EVs from septic mice were slightly smaller (157 ± 2 versus 191 ± 6 nm, p < 0.0001), but more abundant [(1.6 ± 0.14) × 1010 versus (0.93 ± 0.14) × 1010/ml plasma, p < 0.003]. miRNA array revealed that among 65 miRNAs, 8 miRNAs exhibited >1.5-fold increase in septic EVs compared with sham EVs, including miR-126-3p, miR-122-5p, miR-146a-5p, miR-145-5p, miR-26a-5p, miR-150-5p, miR-222-3p, and miR-181a-5p. Septic but not sham EVs were proinflammatory, promoting IL-6, TNF-α, IL-1ß, and MIP-2 production. The effects of EVs were resistant to polymyxin B (an endotoxin inhibitor) but significantly inhibited by anti-miR inhibitors against miR-34a, miR-122, and miR-146a. Moreover, the septic EV-induced cytokine production was attenuated in TLR7-/- or MyD88-/- cells but remained the same in TLR3-/- or Trif-/- cells. In vivo, mice i.p. injected with septic EVs had marked peritoneal neutrophil migration, which was significantly attenuated in MyD88-/- mice. Taken together, these data demonstrate that plasma EVs of septic animals play an important role in inflammation, and EV-associated miRNAs likely mediate the cytokine production via TLR7-MyD88 signaling.

Towards rationally designed biomanufacturing of therapeutic extracellular vesicles: impact of the bioproduction microenvironment.

Extracellular vesicles (EVs), including exosomes, microvesicles, and others, have emerged as potential therapeutics for a variety of applications. Pre-clinical reports of EV efficacy in treatment of non-healing wounds, myocardial infarction, osteoarthritis, traumatic brain injury, spinal cord injury, and many other injuries and diseases demonstrate the versatility of this nascent therapeutic modality. EVs have also been demonstrated to be effective in humans, and clinical trials are underway to further explore their potential. However, for EVs to become a new class of clinical therapeutics, issues related to translation must be addressed. For example, approaches originally developed for cell biomanufacturing, such as hollow fiber bioreactor culture, have been adapted for EV production, but limited knowledge of how the cell culture microenvironment specifically impacts EVs restricts the possibility for rational design and optimization of EV production and potency. In this review, we discuss current knowledge of this issue and delineate potential focus areas for future research towards enabling translation and widespread application of EV-based therapeutics.

Production of Extracellular Vesicles Loaded with Therapeutic Cargo.

Extracellular vesicles (EVs) are biological nanoparticles comprising exosomes, microvesicles, and other heterogeneous nanoscopic vesicle populations that are produced by most cell types. In addition to their putative roles as critical mediators of intercellular communication, EVs have begun to be harnessed as drug delivery vehicles, with early evidence indicating they may have significant advantages over synthetic nanoparticle delivery systems for particular applications. Targeted delivery of EV-encapsulated cargo has already been realized and may have broad applicability; however, methods for producing and purifying EVs and loading them with therapeutic molecules have yet to be standardized. In this chapter, we outline steps for EV isolation and characterization and compare current methods for active and passive loading of EVs with payloads of short interfering RNA (siRNA) or small molecules, with the results revealing that active loading via electroporation increases loading efficiency of siRNA but not of Rhodamine B, a model for a small molecule drug, in HEK293T-derived EVs. The methods described here may inform future design of targeted delivery of nucleic acids or small molecules via EVs.

Ubiquitin Conjugation Probed by Inflammation in Myeloid-Derived Suppressor Cell Extracellular Vesicles.

Ubiquitinated proteins carried by the extracellular vesicles (EV) released by myeloid-derived suppressor cells (MDSC) have been investigated using proteomic strategies to examine the effect of tumor-associated inflammation. EV were collected from MDSC directly following isolation from tumor-bearing mice with low and high inflammation. Among the 1092 proteins (high inflammation) and 925 proteins (low inflammation) identified, more than 50% were observed as ubiquitinated proteoforms. More than three ubiquitin-attachment sites were characterized per ubiquitinated protein, on average. Multiple ubiquitination sites were identified in the pro-inflammatory proteins S100 A8 and S100 A9, characteristic of MDSC and in histones and transcription regulators among other proteins. Spectral counting and pathway analysis suggest that ubiquitination occurs independently of inflammation. Some ubiquitinated proteins were shown to cause the migration of MDSC, which has been previously connected with immune suppression and tumor progression. Finally, MDSC EV are found collectively to carry all the enzymes required to catalyze ubiquitination, and the hypothesis is presented that a portion of the ubiquitinated proteins are produced in situ.

Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs.

Extracellular vesicles (EVs), such as exosomes, have been identified as regulators of vascular remodeling and have promise as therapeutics for vascularization applications. Towards development of EVs as therapeutics, it has been demonstrated that physiological stimuli of angiogenic phenotypes in EV-producing cells can enhance the potency of EVs for vascularization. The goal of this study was to assess whether ethanol, which induces angiogenic phenotypes in endothelial cells, could be employed to enhance endothelial-derived EV vascularization bioactivity. The results indicate that ethanol conditioning of endothelial cells increases the ability of endothelial EVs to induce a pro-vascularization response. This response is due in part to increased CD34 expression in recipient endothelial cells that may result from downregulation of microRNA-106b in EVs isolated from ethanol-conditioned producer endothelial cells. Further, ethanol-induced upregulation of long non-coding RNAs (lncRNAs) HOTAIR and MALAT1 in endothelial EVs was observed to play a significant role in mediating pro-angiogenic effects of these vesicles. Overall, these studies validate ethanol conditioning as a method to enhance the bioactivity of endothelial EVs via regulation of EV-associated microRNAs (miRNAs) and, especially, lncRNAs. Further, the results suggest that alcohol consumption may activate endothelial EVs towards a pro-vascularization phenotype, which could have implications for alcohol-induced tumor angiogenesis.

IL-12 stimulates CTLs to secrete exosomes capable of activating bystander CD8+ T cells.

An effective cytotoxic T lymphocyte (CTL) response against intracellular pathogens is generally accomplished by immense CTL expansion and activation, which can destroy infected cells. Vigorous immune responses can lead to activation of bystander CD8+ T cells, but the contribution from antigen-specific CTLs is not well understood. We found that CTLs secrete extracellular vesicles following antigen stimulation. These CTL-derived vesicles contain CTL proteins and exhibit markers and size profiles consistent with exosomes. Interestingly, further stimulation of CTLs with IL-12 impacts exosome size and leads to selective enrichment of certain exosomal proteins. More important, exosomes from IL-12-stimulated CTLs directly activated bystander naïve CD8+ T cells to produce interferon-γ (IFNγ) and granzyme B (GZB) in the absence of antigens, whereas control exosomes derived from antigen-stimulated CTLs did not. In addition, IL-12 induced exosomes are able to strengthen the effects of weak antigen stimulation on CTLs. Proteomic analysis demonstrates that IL-12 stimulation alters catalytic and binding activities of proteins in CTL exosomes. Our findings indicate that the biological function and morphology of exosomes secreted by CTLs can be influenced by the type of stimulation CTLs receive. Thus, a fully functional, ongoing, antigen-specific CTL response may influence bystander CD8+ T cells through secretion of exosomes.

CD44 Promotes Inflammation and Extracellular Matrix Production During Arteriovenous Fistula Maturation.

OBJECTIVE: Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and poor rates of maturation. We hypothesized that CD44, a widely expressed cellular adhesion molecule that serves as a major receptor for extracellular matrix components, promotes wall thickening and extracellular matrix deposition during AVF maturation. APPROACH AND RESULTS: AVF were created via needle puncture in wild-type C57BL/6J and CD44 knockout mice. CD44 mRNA and protein expression was increased in wild-type AVF. CD44 knockout mice showed no increase in AVF wall thickness (8.9 versus 26.8 µm; P=0.0114), collagen density, and hyaluronic acid density, but similar elastin density when compared with control AVF. CD44 knockout mice also showed no increase in vascular cell adhesion molecule-1 expression, intercellular adhesion molecule-1 expression, and monocyte chemoattractant protein-1 expression in the AVF compared with controls; there were also no increased M2 macrophage markers (transglutaminase-2: 81.5-fold, P=0.0015; interleukin-10: 7.6-fold, P=0.0450) in CD44 knockout mice. Delivery of monocyte chemoattractant protein-1 to CD44 knockout mice rescued the phenotype with thicker AVF walls (27.2 versus 14.7 µm; P=0.0306), increased collagen density (2.4-fold; P=0.0432), and increased number of M2 macrophages (2.1-fold; P=0.0335). CONCLUSIONS: CD44 promotes accumulation of M2 macrophages, extracellular matrix deposition, and wall thickening during AVF maturation. These data show the association of M2 macrophages with wall thickening during AVF maturation and suggest that enhancing CD44 activity may be a strategy to increase AVF maturation.