Harnessing the potential of monocytes/macrophages to regenerate tissue engineered vascular grafts.

Cell-free tissue-engineered vascular grafts provide a promising alternative to treat cardiovascular disease but timely endothelialization is essential for ensuring patency and proper functioning post-implantation. Recent studies from our lab showed that blood cells like monocytes (MCs) and macrophages (Mφ) may contribute directly to cellularization and regeneration of bioengineered arteries in small and large animal models. While MCs and Mφ are leukocytes that are part of the innate immune response, they share common developmental origins with endothelial cells (ECs) and are known to play crucial roles during vessel formation (angiogenesis) and vessel repair after inflammation/injury. They are highly plastic cells that polarize into proinflammatory and anti-inflammatory phenotypes upon exposure to cytokines; and differentiate into other cell types, including EC-like cells, in the presence of appropriate chemical and mechanical stimuli. This review focuses on the developmental origins of MCs and ECs; the role of MCs and Mφ in vessel repair/regeneration during inflammation/injury; and the role of chemical signaling and mechanical forces in Mφ inflammation that mediates vascular graft regeneration. We postulate that comprehensive understanding of these mechanisms will better inform the development of strategies to coax MCs/Mφ into endothelializing the lumen and regenerate the smooth muscle layers of cell-free bioengineered arteries and veins that are designed to treat cardiovascular diseases and perhaps the native vasculature as well.

Altered acetyl-CoA metabolism presents a new potential immunotherapy target in the obese lung microenvironment.

Contrary to the "obesity paradox," which arises from retrospective studies relying on body mass index to define obesity, epidemiologic evidence suggests central or visceral obesity is associated with a higher risk for the development of lung cancer. About 60% of individuals at high risk for developing lung cancer or those already with early-stage disease are either overweight or obese. Findings from resected patient tumors and mouse lung tumor models show obesity dampens immune activity in the tumor microenvironment (TME) encouraging disease progression. In line with this, we have observed a marked, obesity-specific enhancement in the presence and phenotype of immunosuppressive regulatory T (Treg) cells in murine tumors as well as the airways of both humans and mice. Leveraging direct metabolomic measurements and robust inferred analyses from RNA-sequencing data, we here demonstrate for the first time that visceral adiposity alters the lung microenvironment via dysregulated acetyl-CoA metabolism in a direction that facilitates immune suppression and lung carcinogenesis.

Monocyte Recruitment for Vascular Tissue Regeneration.

A strategy to recruit monocytes (MCs) from blood to regenerate vascular tissue from unseeded (cell-free) tissue engineered vascular grafts is presented. When immobilized on the surface of vascular grafts, the fusion protein, H2R5 can capture blood-derived MC under static or flow conditions in a shear stress dependent manner. The bound MC turns into macrophages (Mϕ) expressing both M1 and M2 phenotype specific genes. When H2R5 functionalized acellular-tissue engineered vessels (A-TEVs) are implanted into the mouse aorta, they remain patent and form a continuous endothelium expressing both endothelial cell (EC) and MC specific proteins. Underneath the EC layer, multiple cells layers are formed coexpressing both smooth muscle cell (SMC) and MC specific markers. Lineage tracing analysis using a novel CX3CR1-confetti mouse model demonstrates that fluorescently labeled MC populates the graft lumen by two and four weeks postimplantation, providing direct evidence in support of MC/Mϕ recruitment to the graft lumen. Given their abundance in the blood, circulating MCs may be a great source of cells that contribute directly to the endothelialization and vascular wall formation of acellular vascular grafts under the right chemical and biomechanical cues.

Generating Monocyte-Derived Endothelial-like Cells for Vascular Regeneration.

A major limitation in engineering vascular grafts is the lack of proper endothelium to prevent thrombosis and subsequent graft failure. Obtaining endothelial cells from patients' vasculature is intrusive and requires extensive culture time. Here we present an alternative strategy wherein abundant and easily accessible monocytes from peripheral blood are cultured and differentiated towards an endothelial-like state capable of preventing thrombosis through production of nitric oxide and formation of endothelial adherens junctions. Considering the plethora of monocytes present within peripheral blood, this method provides a robust alternative to generating endothelial cells required for vascular graft production.

Visceral Obesity Promotes Lung Cancer Progression-Toward Resolution of the Obesity Paradox in Lung Cancer.

INTRODUCTION: Although obesity is associated with adverse cancer outcomes in general, most retrospective clinical studies suggest a beneficial effect of obesity in NSCLC. METHODS: Hypothesizing that this "obesity paradox" arises partly from the limitations of using body mass index (BMI) to measure obesity, we quantified adiposity using preoperative computed tomography images. This allowed the specific determination of central obesity as abdominal visceral fat area normalized to total fat area (visceral fat index [VFI]). In addition, owing to the previously reported salutary effect of metformin on high-BMI patients with lung cancer, metformin users were excluded. We then explored associations between visceral obesity and outcomes after surgical resection of stage I and II NSCLC. We also explored potential immunologic underpinnings of such association using complimentary analyses of tumor gene expression data from NSCLC tumors and the tumor transcriptome and immune microenvironment in an immunocompetent model of lung cancer with diet-induced obesity. RESULTS: We found that in 513 patients with stage I and II NSCLC undergoing lobectomy, a high VFI is associated with decreased recurrence-free and overall survival. VFI was also inversely related to an inflammatory transcriptomic signature in NSCLC tumors, consistent with observations made in immunocompetent murine models wherein diet-induced obesity promoted cancer progression while exacerbating elements of immune suppression in the tumor niche. CONCLUSIONS: In all, this study uses multiple lines of evidence to reveal the adverse effects of visceral obesity in patients with NSCLC, which align with those found in animal models. Thus, the obesity paradox may, at least in part, be secondary to the use of BMI as a measure of obesity and the confounding effects of metformin use.

Endothelialization of arterial vascular grafts by circulating monocytes.

Recently our group demonstrated that acellular tissue engineered vessels (A-TEVs) comprised of small intestinal submucosa (SIS) immobilized with heparin and vascular endothelial growth factor (VEGF) could be implanted into the arterial system of a pre-clinical ovine animal model, where they endothelialized within one month and remained patent. Here we report that immobilized VEGF captures blood circulating monocytes (MC) with high specificity under a range of shear stresses. Adherent MC differentiate into a mixed endothelial (EC) and macrophage (Mφ) phenotype and further develop into mature EC that align in the direction of flow and produce nitric oxide under high shear stress. In-vivo, newly recruited cells on the vascular lumen express MC markers and at later times they co-express MC and EC-specific proteins and maintain graft patency. This novel finding indicates that the highly prevalent circulating MC contribute directly to the endothelialization of acellular vascular grafts under the right chemical and biomechanical cues.

Cell-free vascular grafts that grow with the host.

Cell-free small diameter vascular grafts, based on small intestinal submucosa (SIS) functionalized with heparin and vascular endothelial growth factor (VEGF) manufactured and implanted successfully into the arterial system of neonatal lambs, where they remained patent and grew in size with the host to a similar extent and with similar rate as native arteries. Acellular tissue engineered vessels (A-TEV) integrated seamlessly into the native vasculature and developed confluent, functional endothelium that afforded patency. The medial layer was infiltrated by smooth muscle cells, showed no signs of calcification and developed contractile function. The vascular wall underwent remarkable extracellular matrix remodeling exhibiting elastin fibers and even inner elastic lamina within six months. Taken together, our results suggest that VEGF-based A-TEVs may be suitable for treatment of congenital heart disorders to alleviate the need for repeated surgeries, which are currently standard practice.

Antigen delivery format variation and formulation stability through use of a hybrid vector.

A hybrid biological-biomaterial antigen delivery vector comprised of a polymeric shell encapsulating an Escherichia coli core was previously developed for in situ antigen production and subsequent delivery. Due to the engineering capacity of the bacterial core, the hybrid vector provides unique opportunities for immunogenicity optimization through varying cellular localization (cytoplasm, periplasm, cellular surface) and type (protein or DNA) of antigen. In this work, three protein-based hybrid vector formats were compared in which the pneumococcal surface protein A (PspA) was localized to the cytoplasm, surface, and periplasmic space of the bacterial core for vaccination against pneumococcal disease. Furthermore, we tested the hybrid vector's capacity as a DNA vaccine against Streptococcus pneumoniae by introducing a plasmid into the bacterial core to facilitate PspA expression in antigen presenting cells (APCs). Through testing these various formulations, we determined that cytoplasmic accumulation of PspA elicited the strongest immune response (antibody production and protection against bacterial challenge) and enabled complete protection at substantially lower doses when compared to vaccination with PspA + adjuvant. We also improved the storage stability of the hybrid vector to retain complete activity after 1 month at 4 °C using an approach in which hybrid vectors suspended in a microbial freeze drying buffer were desiccated. These results demonstrate the flexibility and robustness of the hybrid vector formulation, which has the potential to be a potent vaccine against S. pneumoniae.

Synergistic effects of laminin-1 peptides, VEGF and FGF9 on salivary gland regeneration.

Hyposalivation is associated with radiation therapy, Sjögren's syndrome and/or aging, and is a significant clinical problem that decreases oral health and overall health in many patients and currently lacks effective treatment. Hence, methods to regenerate salivary glands and restore saliva secretion are urgently needed. To this end, this study describes the modification of fibrin hydrogels with a combination of laminin-1 peptides (YIGSR and A99) and human growth factors (vascular endothelial growth factor and fibroblast growth factor 9) to enhance regeneration in a salivary gland injury mouse model. Our results indicate that these fortified hydrogels enhanced angiogenesis and neurogenesis while promoting formation of acinar structures, thereby leading to enhanced saliva secretion. Such functional recovery indicates salivary gland regeneration and suggests that our technology may be useful in promoting gland regeneration and reversing hyposalivation in a clinical setting. STATEMENT OF SIGNIFICANCE: We engineered Fibrin Hydrogels (FH) to contain multiple regenerative cues including laminin-1 peptides (L1p) and growth factors (GFs). L1p and GF modified FH were used to induce salivary gland regeneration in a wounded mouse model. Treatment with L1p and GF modified FH promoted salivary epithelial tissue regeneration, vascularization, neurogenesis and healing as compared to L1p-FH or FH alone. Results indicate that L1p and GF modified FH can be used for future therapeutic applications.

Implantation of VEGF-functionalized cell-free vascular grafts: regenerative and immunological response.

Recently, our group demonstrated that immobilized VEGF can capture flowing endothelial cells (ECs) from the blood in vitro and promote endothelialization and patency of acellular tissue-engineered vessels (A-TEVs) into the arterial system of an ovine animal model. Here, we demonstrate implantability of submillimeter diameter heparin and VEGF-decorated A-TEVs in a mouse model and discuss the cellular and immunologic response. At 1 mo postimplantation, the graft lumen was fully endothelialized, as shown by expression of EC markers such as CD144, eNOS, CD31, and VEGFR2. Interestingly, the same cells coexpressed leukocyte/macrophage (Mϕ) markers CD14, CD16, VEGFR1, CD38, and EGR2. Notably, there was a stark difference in the cellular makeup between grafts containing VEGF and those containing heparin alone. In VEGF-containing grafts, infiltrating monocytes (MCs) converted into anti-inflammatory M2-Mϕs, and the grafts developed well-demarcated luminal and medial layers resembling those of native arteries. In contrast, in grafts containing only heparin, MCs converted primarily into M1-Mϕs, and the endothelial and smooth muscle layers were not well defined. Our results indicate that VEGF may play an important role in regulating A-TEV patency and regeneration, possibly by regulating the inflammatory response to the implants.-Smith, R. J., Jr., Yi, T., Nasiri, B., Breuer, C. K., Andreadis, S. T. Implantation of VEGF-functionalized cell-free vascular grafts: regenerative and immunological response.

In situ pneumococcal vaccine production and delivery through a hybrid biological-biomaterial vector.

The type and potency of an immune response provoked during vaccination will determine ultimate success in disease prevention. The basis for this response will be the design and implementation of antigen presentation to the immune system. Whereas direct antigen administration will elicit some form of immunological response, a more sophisticated approach would couple the antigen of interest to a vector capable of broad delivery formats and designed for heightened response. New antigens associated with pneumococcal disease virulence were used to test the delivery and adjuvant capabilities of a hybrid biological-biomaterial vector consisting of a bacterial core electrostatically coated with a cationic polymer. The hybrid design provides (i) passive and active targeting of antigen-presenting cells, (ii) natural and multicomponent adjuvant properties, (iii) dual intracellular delivery mechanisms, and (iv) a simple formulation mechanism. In addition, the hybrid format enables device-specific, or in situ, antigen production and consolidation via localization within the bacterial component of the vector. This capability eliminates the need for dedicated antigen production and purification before vaccination efforts while leveraging the aforementioned features of the overall delivery device. We present the first disease-specific utilization of the vector toward pneumococcal disease highlighted by improved immune responses and protective capabilities when tested against traditional vaccine formulations and a range of clinically relevant Streptococcus pneumoniae strains. More broadly, the results point to similar levels of success with other diseases that would benefit from the production, delivery, and efficacy capabilities offered by the hybrid vector.

Hybrid Biomaterial with Conjugated Growth Factors and Mesenchymal Stem Cells for Ectopic Bone Formation.

Bone is a highly vascularized tissue and efficient bone regeneration requires neovascularization, especially for critical-sized bone defects. We developed a novel hybrid biomaterial comprising nanocalcium sulfate (nCS) and fibrin hydrogel to deliver mesenchymal stem cells (MSCs) and angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 9 (FGF9), to promote neovascularization and bone formation. MSC and growth factor(s)-loaded scaffolds were implanted subcutaneously into mice to examine their angiogenic and osteogenic potential. Micro CT, alkaline phosphatase activity assay, and histological analysis were used to evaluate bone formation, while immunohistochemistry was employed to assess neovessel formation. The presence of fibrin preserved the nCS scaffold structure and promoted de novo bone formation. In addition, the presence of bone morphogenic protein 2-expressing MSC in nCS and fibrin hydrogels improved bone regeneration significantly. While FGF9 alone had no significant effect, the combination FGF9 and VEGF conjugated in fibrin enhanced neovascularization and bone formation more than 4-fold compared to nCS with MSC. Overall, our results suggested that the combination of nCS (to support bone formation) with a fibrin-based VEGF/FGF9 release system (support vascular formation) is an innovative and effective strategy that significantly enhanced ectopic bone formation in vivo.

Successful endothelialization and remodeling of a cell-free small-diameter arterial graft in a large animal model.

The large number of coronary artery bypass procedures necessitates development of off-the-shelf vascular grafts that do not require cell or tissue harvest from patients. However, immediate thrombus formation after implantation due to the absence of a healthy endothelium is very likely. Here we present the successful development of an acellular tissue engineered vessel (A-TEV) based on small intestinal submucosa that was functionalized sequentially with heparin and VEGF. A-TEVs were implanted into the carotid artery of an ovine model demonstrating high patency rates and significant host cell infiltration as early as one week post-implantation. At one month, a confluent and functional endothelium was present and the vascular wall showed significant infiltration of host smooth muscle cells exhibiting vascular contractility in response to vaso-agonists. After three months, the endothelium aligned in the direction of flow and the medial layer comprised of circumferentially aligned smooth muscle cells. A-TEVs demonstrated high elastin and collagen content as well as impressive mechanical properties and vascular contractility comparable to native arteries. This is the first demonstration of successful endothelialization, remodeling, and development of vascular function of a cell-free vascular graft that was implanted in the arterial circulation of a pre-clinical animal model.

Capture of endothelial cells under flow using immobilized vascular endothelial growth factor.

We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-l-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm(2)) to physiological (15 dyne/cm(2)). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC.

Airborne fiber size characterization in exposure estimation: Evaluation of a modified transmission electron microcopy protocol for asbestos and potential use for carbon nanotubes and nanofibers.

BACKGROUND: Airborne fiber size has been shown to be an important factor relative to adverse lung effects of asbestos and suggested in animal studies of carbon nanotubes and nanofibers (CNT/CNF). MATERIALS AND METHODS: The International Standards Organization (ISO) transmission electron microscopy (TEM) method for asbestos was modified to increase the statistical precision of fiber size determinations, improve efficiency, and reduce analysis costs. Comparisons of the fiber size distributions and exposure indices by laboratory and counting method were performed. RESULTS: No significant differences in size distributions by the ISO and modified ISO methods were observed. Small but statistically-significant inter-lab differences in the proportion of fibers in some size bins were found, but these differences had little impact on the summary exposure indices. The modified ISO method produced slightly more precise estimates of the long fiber fraction (>15 µm). CONCLUSIONS: The modified ISO method may be useful for estimating size-specific structure exposures, including CNT/CNF, for risk assessment research.

Contributions of dust exposure and cigarette smoking to emphysema severity in coal miners in the United States.

RATIONALE: Previous studies have shown associations between dust exposure or lung burden and emphysema in coal miners, although the separate contributions of various predictors have not been clearly demonstrated. OBJECTIVES: To quantitatively evaluate the relationship between cumulative exposure to respirable coal mine dust, cigarette smoking, and other factors on emphysema severity. METHODS: The study group included 722 autopsied coal miners and nonminers in the United States. Data on work history, smoking, race, and age at death were obtained from medical records and questionnaire completed by next-of-kin. Emphysema was classified and graded using a standardized schema. Job-specific mean concentrations of respirable coal mine dust were matched with work histories to estimate cumulative exposure. Relationships between various metrics of dust exposure (including cumulative exposure and lung dust burden) and emphysema severity were investigated in weighted least squares regression models. MEASUREMENTS AND MAIN RESULTS: Emphysema severity was significantly elevated in coal miners compared with nonminers among ever- and never-smokers (P < 0.0001). Cumulative exposure to respirable coal mine dust or coal dust retained in the lungs were significant predictors of emphysema severity (P < 0.0001) after accounting for cigarette smoking, age at death, and race. The contributions of coal mine dust exposure and cigarette smoking were similar in predicting emphysema severity averaged over this cohort. CONCLUSIONS: Coal dust exposure, cigarette smoking, age, and race are significant and additive predictors of emphysema severity in this study.

Hexavalent chromium and lung cancer in the chromate industry: a quantitative risk assessment.

The purpose of this investigation was to estimate excess lifetime risk of lung cancer death resulting from occupational exposure to hexavalent-chromium-containing dusts and mists. The mortality experience in a previously studied cohort of 2,357 chromate chemical production workers with 122 lung cancer deaths was analyzed with Poisson regression methods. Extensive records of air samples evaluated for water-soluble total hexavalent chromium were available for the entire employment history of this cohort. Six different models of exposure-response for hexavalent chromium were evaluated by comparing deviances and inspection of cubic splines. Smoking (pack-years) imputed from cigarette use at hire was included in the model. Lifetime risks of lung cancer death from exposure to hexavalent chromium (assuming up to 45 years of exposure) were estimated using an actuarial calculation that accounts for competing causes of death. A linear relative rate model gave a good and readily interpretable fit to the data. The estimated rate ratio for 1 mg/m3-yr of cumulative exposure to hexavalent chromium (as CrO3), with a lag of five years, was RR=2.44 (95% CI=1.54-3.83). The excess lifetime risk of lung cancer death from exposure to hexavalent chromium at the current OSHA permissible exposure limit (PEL) (0.10 mg/m3) was estimated to be 255 per 1,000 (95% CI: 109-416). This estimate is comparable to previous estimates by U.S. EPA, California EPA, and OSHA using different occupational data. Our analysis predicts that current occupational standards for hexavalent chromium permit a lifetime excess risk of dying of lung cancer that exceeds 1 in 10, which is consistent with previous risk assessments.

Evaluation of the risk of noise-induced hearing loss among unscreened male industrial workers.

Variability in background risk and distribution of various risk factors for hearing loss may explain some of the diversity in excess risk of noise-induced hearing loss (NIHL). This paper examines the impact of various risk factors on excess risk estimates of NIHL using data from the 1968-1972 NIOSH Occupational Noise and Hearing Survey (ONHS). Previous analyses of a subset of these data focused on 1172 highly "screened" workers. In the current analysis, an additional 894 white males (609 noise-exposed and 285 controls), who were excluded for various reasons (i.e., nonoccupational noise exposure, otologic or medical conditions affecting hearing, prior occupational noise exposure) have been added 2066) to assess excess risk of noise-induced material impairment in an unscreened population. Data are analyzed by age, duration of exposure, and sound level (8-h TWA) for four different definitions of noise-induced hearing impairment, defined as the binaural pure-tone average (PTA) hearing threshold level greater than 25 dB for the following frequencies: (a) 1-4 kHz (PTA1234), (b) 1-3 kHz (PTA123), (c) 0.5, 1, and 2 kHz (PTA512), and (d) 3, 4, and 6 kHz (PTA346). Results indicate that populations with higher background risks of hearing loss may show lower excess risks attributable to noise relative to highly screened populations. Estimates of lifetime excess risk of hearing impairment were found to be significantly different between screened and unscreened population for noise levels greater than 90 dBA. Predicted age-related risk of material hearing impairment in the ONHS unscreened population was similar to that predicted from Annex B and C of ANSI S3.44 for ages less than 60 years. Results underscore the importance of understanding differential risk patterns for hearing loss and the use of appropriate reference (control) populations when evaluating risk of noise-induced hearing impairment among contemporary industrial populations.

Validation of injection parameters for catheter-directed intraarterial gadolinium-enhanced MR angiography.

RATIONALE AND OBJECTIVES: Catheter-directed intraarterial (IA) injections of gadolinium contrast agents may be used during endovascular interventions with magnetic resonance (MR) imaging guidance. Injection protocols require further validation. Using a flow phantom and swine, the authors aimed to (a) measure the optimal arterial gadolinium concentration ([Gd]) required for MR angiography and (b) validate a proposed IA injection protocol for gadolinium-enhanced MR angiography. MATERIALS AND METHODS: For in vitro experiments, the authors placed a catheter in the aorta of an aorto-renal-iliac flow phantom. Injected [Gd], injection rates, and aortic blood flow rates were varied independently for 36 separate IA gadolinium injections. The authors performed 2D and 3D MR angiography with a fast spoiled gradient-recalled echo sequence. For subsequent in vivo experiments, they selectively placed catheters within the aorta, renal artery, or common iliac artery of three pigs. Injection rate and injected [Gd] were varied. The authors performed 32 separate IA gadolinium injections for 2D MR angiography. Signal-to-noise ratios (SNRs) were compared for the various combinations of injection rate and injected [Gd]. RESULTS: In vitro, an arterial [Gd] of 2%-4% produced an optimal SNR for 2D MR angiography, and 3%-5% was best for 3D MR angiography. In swine, an arterial [Gd] of 1%-4% produced an optimal SNR. In the phantom and swine experiments, SNR was maintained at higher injection rates by inversely varying the injected [Gd]. CONCLUSION: Dilute arterial [Gd] is required for optimal IA gadolinium-enhanced MR angiography. To maintain an optimal SNR, injection rates and injected [Gd] should be varied inversely. The postulated injection protocol was validated.