Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy.

The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-ß superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.

Extracellular vesicles in sepsis plasma mediate neuronal inflammation in the brain through miRNAs and innate immune signaling.

BACKGROUND: Neuroinflammation reportedly plays a critical role in the pathogenesis of sepsis-associated encephalopathy (SAE). We previously reported that circulating plasma extracellular vesicles (EVs) from septic mice are proinflammatory. In the current study, we tested the role of sepsis plasma EVs in neuroinflammation. METHODS: To track EVs in cells and tissues, HEK293T cell-derived EVs were labeled with the fluorescent dye PKH26. Cecal ligation and puncture (CLP) was conducted to model polymicrobial sepsis in mice. Plasma EVs were isolated by ultracentrifugation and their role in promoting neuronal inflammation was tested following intracerebroventricular (ICV) injection. miRNA inhibitors (anti-miR-146a, -122, -34a, and -145a) were applied to determine the effects of EV cargo miRNAs in the brain. A cytokine array was performed to profile microglia-released protein mediators. TLR7- or MyD88-knockout (KO) mice were utilized to determine the underlying mechanism of EVs-mediated neuroinflammation. RESULTS: We observed the uptake of fluorescent PKH26-EVs inside the cell bodies of both microglia and neurons. Sepsis plasma EVs led to a dose-dependent cytokine release in cultured microglia, which was partially attenuated by miRNA inhibitors against the target miRNAs and in TLR7-KO cells. When administered via the ICV, sepsis plasma EVs resulted in a marked increase in the accumulation of innate immune cells, including monocyte and neutrophil and cytokine gene expression, in the brain. Although sepsis plasma EVs had no direct effect on cytokine production or neuronal injury in vitro, the conditioned media (CM) of microglia treated with sepsis plasma EVs induced neuronal cell death as evidenced by increased caspase-3 cleavage and Annexin-V staining. Cytokine arrays and bioinformatics analysis of the microglial CM revealed multiple cytokines/chemokines and other factors functionally linked to leukocyte chemotaxis and migration, TLR signaling, and neuronal death. Moreover, sepsis plasma EV-induced brain inflammation in vivo was significantly dependent on MyD88. CONCLUSIONS: Circulating plasma EVs in septic mice cause a microglial proinflammatory response in vitro and a brain innate immune response in vivo, some of which are in part mediated by TLR7 in vitro and MyD88 signaling in vivo. These findings highlight the importance of circulating EVs in brain inflammation during sepsis.

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination.

Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a human embryonic kidney cells (HEK293T) EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static versus dynamic) and isolation techniques (i.e., ultracentrifugation versus tangential flow filtration versus size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established lipopolysaccharide-stimulated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., interleukin-6, RANTES [regulated upon activation, normal T cell expressed and secreted]) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to non-EV fetal bovine serum components in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.

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.

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.

Chitosan Particles Complexed with CA5-HIF-1α Plasmids Increase Angiogenesis and Improve Wound Healing.

Wound therapies involving gene delivery to the skin have significant potential due to the advantage and ease of local treatment. However, choosing the appropriate vector to enable successful gene expression while also ensuring that the treatment's immediate material components are conducive to healing itself is critical. In this study, we utilized a particulate formulation of the polymer chitosan (chitosan particles, CPs) as a non-viral vector for the delivery of a plasmid encoding human CA5-HIF-1α, a degradation resistant form of HIF-1α, to enhance wound healing. We also compared the angiogenic potential of our treatment (HIF/CPs) to that of chitosan particles containing only the plasmid backbone (bb/CPs) and the chitosan particle vector alone (CPs). Our results indicate that chitosan particles exert angiogenic effects that are enhanced with the human CA5-HIF-1α-encoded plasmid. Moreover, HIF/CPs enhanced wound healing in diabetic db/db mice (p < 0.01), and healed tissue was found to contain a significantly increased number of blood vessels compared to bb/CPs (p < 0.01), CPs (p < 0.05) and no-treatment groups (p < 0.01). Thus, this study represents a method of gene delivery to the skin that utilizes an inherently pro-wound-healing polymer as a vector for plasmid DNA that has broad application for the expression of other therapeutic genes.

Enhanced Loading of Functional miRNA Cargo via pH Gradient Modification of Extracellular Vesicles.

Based on their identification as physiological nucleic acid carriers in humans and other organisms, extracellular vesicles (EVs) have been explored as therapeutic delivery vehicles for DNA, RNA, and other cargo. However, efficient loading and functional delivery of nucleic acids remain a challenge, largely because of potential sources of degradation and aggregation. Here, we report that protonation of EVs to generate a pH gradient across EV membranes can be utilized to enhance vesicle loading of nucleic acid cargo, specifically microRNA (miRNA), small interfering RNA (siRNA), and single-stranded DNA (ssDNA). The loading process did not impair cellular uptake of EVs, nor did it promote any significant EV-induced toxicity response in mice. Cargo functionality was verified by loading HEK293T EVs with either pro- or anti-inflammatory miRNAs and observing the effective regulation of corresponding cellular cytokine levels. Critically, this loading increase is comparable with what can be accomplished by methods such as sonication and electroporation, and is achievable without the introduction of energy associated with these methods that can potentially damage labile nucleic acid cargo.

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.

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.

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.

Engineered Multivalency Enhances Affibody-Based HER3 Inhibition and Downregulation in Cancer Cells.

The receptor tyrosine kinase HER3 has emerged as a therapeutic target in ovarian, prostate, breast, lung, and other cancers due to its ability to potently activate the PI3K/Akt pathway, especially via dimerization with HER2, as well as for its role in mediating drug resistance. Enhanced efficacy of HER3-targeted therapeutics would therefore benefit a wide range of patients. This study evaluated the potential of multivalent presentation, through protein engineering, to enhance the effectiveness of HER3-targeted affibodies as alternatives to monoclonal antibody therapeutics. Assessment of multivalent affibodies on a variety of cancer cell lines revealed their broad ability to improve inhibition of Neuregulin (NRG)-induced HER3 and Akt phosphorylation compared to monovalent analogues. Engineered multivalency also promoted enhanced cancer cell growth inhibition by affibodies as single agents and as part of combination therapy approaches. Mechanistic investigations revealed that engineered multivalency enhanced affibody-mediated HER3 downregulation in multiple cancer cell types. Overall, these results highlight the promise of engineered multivalency as a general strategy for enhanced efficacy of HER3-targeted therapeutics against a variety of cancers.

Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.

Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

Protein engineering for cardiovascular therapeutics: untapped potential for cardiac repair.

A number of new and innovative approaches for repairing damaged myocardium are currently undergoing investigation, with several encouraging results. In addition to the progression of stem cell-based approaches and gene therapy/silencing methods, evidence continues to emerge that protein therapeutics may be used to directly promote cardiac repair and even regeneration. However, proteins are often limited in their therapeutic potential by short local half-lives and insufficient bioavailability and bioactivity, and many academic laboratories studying cardiovascular diseases are more comfortable with molecular and cellular biology than with protein biochemistry. Protein engineering has been used broadly to overcome weaknesses traditionally associated with protein therapeutics and has the potential to specifically enhance the efficacy of molecules for cardiac repair. However, protein engineering as a strategy has not yet been used in the development of cardiovascular therapeutics to the degree that it has been used in other fields. In this review, we discuss the role of engineered proteins in cardiovascular therapies to date. Further, we address the promise of applying emerging protein engineering technologies to cardiovascular medicine and the barriers that must be overcome to enable the ultimate success of this approach.

An engineered bivalent neuregulin protects against doxorubicin-induced cardiotoxicity with reduced proneoplastic potential.

BACKGROUND: Doxorubicin (DOXO) is an effective anthracycline chemotherapeutic, but its use is limited by cumulative dose-dependent cardiotoxicity. Neuregulin-1ß is an ErbB receptor family ligand that is effective against DOXO-induced cardiomyopathy in experimental models but is also proneoplastic. We previously showed that an engineered bivalent neuregulin-1ß (NN) has reduced proneoplastic potential in comparison with the epidermal growth factor-like domain of neuregulin-1ß (NRG), an effect mediated by receptor biasing toward ErbB3 homotypic interactions uncommonly formed by native neuregulin-1ß. Here, we hypothesized that a newly formulated, covalent NN would be cardioprotective with reduced proneoplastic effects in comparison with NRG. METHODS AND RESULTS: NN was expressed as a maltose-binding protein fusion in Escherichia coli. As established previously, NN stimulated antineoplastic or cytostatic signaling and phenotype in cancer cells, whereas NRG stimulated proneoplastic signaling and phenotype. In neonatal rat cardiomyocytes, NN and NRG induced similar downstream signaling. NN, like NRG, attenuated the double-stranded DNA breaks associated with DOXO exposure in neonatal rat cardiomyocytes and human cardiomyocytes derived from induced pluripotent stem cells. NN treatment significantly attenuated DOXO-induced decrease in fractional shortening as measured by blinded echocardiography in mice in a chronic cardiomyopathy model (57.7±0.6% versus 50.9±2.6%, P=0.004), whereas native NRG had no significant effect (49.4±3.7% versus 50.9±2.6%, P=0.813). CONCLUSIONS: NN is a cardioprotective agent that promotes cardiomyocyte survival and improves cardiac function in DOXO-induced cardiotoxicity. Given the reduced proneoplastic potential of NN versus NRG, NN has translational potential for cardioprotection in patients with cancer receiving anthracyclines.

Analysis of Bacterial Extracellular Vesicles by Immunogold Transmission Electron Microscopy.

Bacterial extracellular vesicles (BEVs) have emerged as mediators of transkingdom communication with numerous potential biotechnological applications. As such, investigation of BEV's protein composition holds promise to uncover new biological mechanisms, such as in microbiome-host communication or pathogen infection. Additionally, bioengineering of BEV protein composition can enhance their therapeutic potential. However, accurate assessment of BEV protein cargo is limited by their nanometer size, which precludes light microscopy imaging, as well as by co-isolation of protein impurities during separation processes. A solution to these challenges is found in immunogold transmission electron microscopy (TEM), which combines antibody-based labeling with direct visualization of BEVs. Several challenges are commonly encountered during immunogold TEM analysis of BEVs, most notably inefficient antibody labeling and poor contrast. Here, we present an optimized protocol for immunogold TEM analysis of BEVs that overcomes such challenges.

Extracellular vesicles as therapeutics for inflammation and infection.

Extracellular vesicles (EVs) are an emergent next-generation biotechnology with broad application potential. In particular, immunomodulatory bioactivity of EVs leading to anti-inflammatory effects is well-characterized. Cell source and culture conditions are critical determinants of EV therapeutic efficacy, while augmenting EV anti-inflammatory bioactivity via diverse strategies, including RNA cargo loading and protein surface display, has proven effective. Yet, translational challenges remain. Additionally, the potential of direct antimicrobial EV functionality has only recently emerged but offers the possibility of overcoming drug-resistant bacterial and fungal infections through novel, multifactorial mechanisms. As discussed herein, these application areas are brought together by the potential for synergistic benefit from technological developments related to EV cargo loading and biomanufacturing.

Separation of Bacterial Extracellular Vesicles via High-Performance Anion Exchange Chromatography.

Bacterial extracellular vesicles (BEVs) have extraordinary biotechnological potential, but traditional purification methods lack desirable scalability and commonly co-isolate protein impurities, limiting clinical translation. Anion exchange chromatography (AEC) separates molecules based on differences in net charge and is widely used for industrial biomanufacturing of protein therapeutics. Recently, AEC has recently been applied for purification of EVs from both mammalian and bacterial sources. Since most bacteria produce BEVs with a negative surface membrane change, AEC can potentially be widely used for BEV purification. Here, we describe a method utilizing high-performance AEC (HPAEC) in tandem with size-based tangential flow filtration for improved BEV purification. We have previously found this method can reduce co-isolated protein impurities and potentiate anti-inflammatory bioactivity of probiotic BEVs. Thus, this method holds promise as a scalable alternative for improved BEV purification.

Enhanced Extracellular Vesicle Cargo Loading via microRNA Biogenesis Pathway Modulation.

Extracellular vesicles (EVs) are physiological vectors for the intercellular transport of a variety of molecules. Among these, small RNAs, and especially microRNAs (miRNAs), have been identified as prevalent components, and there has thus been a robust investigation of EVs for therapeutic miRNAs delivery. However, intrinsic levels of EV-associated miRNAs are generally too low to enable efficient and effective therapeutic outcomes. We hypothesized that miRNA localization to EVs could be improved by limiting competing interactions that occur throughout the miRNA biogenesis process. Using miR-146a-5p as a model, modulation of transcription, nuclear export, and enzymatic cleavage steps of miRNA biogenesis were tested for impact on EV miRNA loading. Working in HEK293T cells, various alterations in the EV biogenesis pathway were shown to impact miRNA localization to EVs. The system was then applied in induced pluripotent stem cells (iPSCs), a more promising substrate for therapeutic EV production, and EVs were separated and assessed for anti-inflammatory efficacy in vitro and in a murine colitis model, where the preservation of function was validated. Overall, the results highlight necessary considerations when designing a cell culture system for the devoted production of miRNA-loaded EVs.

Combination delivery of TGF-ß inhibitor and IL-2 by nanoscale liposomal polymeric gels enhances tumour immunotherapy.

The tumour microenvironment thwarts conventional immunotherapy through multiple immunologic mechanisms, such as the secretion of the transforming growth factor-ß (TGF-ß), which stunts local tumour immune responses. Therefore, high doses of interleukin-2 (IL-2), a conventional cytokine for metastatic melanoma, induces only limited responses. To overcome the immunoinhibitory nature of the tumour microenvironment, we developed nanoscale liposomal polymeric gels (nanolipogels; nLGs) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers that can deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines in a sustained fashion to the tumour microenvironment. nLGs releasing TGF-ß inhibitor and IL-2 significantly delayed tumour growth, increased survival of tumour-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8(+) T-cell infiltration. We demonstrate that the efficacy of nLGs in tumour immunotherapy results from a crucial mechanism involving activation of both innate and adaptive immune responses.

Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling.

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.