Exposure of Induced Pluripotent Stem Cell-Derived Vascular Endothelial and Smooth Muscle Cells in Coculture to Hemodynamics Induces Primary Vascular Cell-Like Phenotypes.

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial (iEC) and smooth muscle (iSMC) cells. However, because iECs and iSMCs are not derived from an intact blood vessel, they represent an immature phenotype. Hemodynamics and heterotypic cell:cell communication play important roles in vascular cell phenotypic modulation. Here we tested the hypothesis that hemodynamic exposure of iECs in coculture with iSMCs induces an in vivo-like phenotype. iECs and iSMCs were cocultured under vascular region-specific blood flow hemodynamics, and compared to hemodynamic cocultures of blood vessel-derived endothelial (pEC) and smooth muscle (pSMC) cells. Hemodynamic flow-induced gene expression positively correlated between pECs and iECs as well as pSMCs and iSMCs. While endothelial nitric oxide synthase 3 protein was lower in iECs than pECs, iECs were functionally mature as seen by acetylated-low-density lipoprotein (LDL) uptake. SMC contractile protein markers were also positively correlated between pSMCs and iSMCs. Exposure of iECs and pECs to atheroprone hemodynamics with oxidized-LDL induced an inflammatory response in both. Dysfunction of the transforming growth factor ß (TGFß) pathway is seen in several vascular diseases, and iECs and iSMCs exhibited a transcriptomic prolife similar to pECs and pSMCs, respectively, in their responses to LY2109761-mediated transforming growth factor ß receptor I/II (TGFßRI/II) inhibition. Although there are differences between ECs and SMCs derived from iPSCs versus blood vessels, hemodynamic coculture restores a high degree of similarity in their responses to pathological stimuli associated with vascular diseases. Thus, iPSC-derived vascular cells exposed to hemodynamics may provide a viable system for modeling rare vascular diseases and testing new therapeutic approaches. Stem Cells Translational Medicine 2017;6:1673-1683.

Transcriptional profiling suggests that Nevirapine and Ritonavir cause drug induced liver injury through distinct mechanisms in primary human hepatocytes.

Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to concentrations approximating clinical therapeutic and supra-therapeutic levels of Nevirapine (11.3 and 175.0 µM) and Ritonavir (3.5 and 62.4 µM) for 48 h. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain with simultaneous mitochondrial uncoupling supporting clinical reports of a lower propensity for steatosis. This in vitro study offers insights into the disparate direct and immune-mediated toxicity mechanisms underlying Nevirapine and Ritonavir toxicity in the clinic.

Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related Atherosclerosis.

BACKGROUND: High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). METHODS AND RESULTS: A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC(-/-) mice. In RBCs from HFD-fed wild-type and DARC(-/-) mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. CONCLUSIONS: RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.

Endovascular coil embolization of a spinal epidural arteriovenous fistula with associated cord compression from an enlarging venous varix.

Spinal arteriovenous fistulas (AVFs) completely isolated to the epidural compartment are exceedingly rare. As such, the optimal management of these lesions is poorly defined. The aim of this technical note is to describe our endovascular technique for the occlusion of a purely epidural AVF of the thoracic spine associated with cord compression from an associated enlarging venous varix. A 40-year-old male presented with severe right-sided back pain and anterior thigh numbness after a sports-related back injury six months previously. Spinal magnetic resonance imaging (MRI) showed an enhancing, extradural mass lesion at T12. Spinal angiography revealed an epidural AVF supplied by a radicular branch of the right T12 subcostal artery and draining into the paravertebral lumbar veins, as well as an adjacent 20 × 13 mm(2) contrast-filling sac, compatible with a dilated venous varix. There was no evidence of intradural venous drainage. We elected to proceed with endovascular treatment of the lesion. At the time of embolization five days later, the venous varix had enlarged to 26 × 16 mm(2). The T12 epidural AVF was completely occluded with two coils, without residual or recurrent AVF on follow-up angiography one month later. The patient made a full recovery, and complete resolution of the venous varix and cord compression were noted on MRI at three months follow-up. Endovascular coil embolization can be successfully employed for the treatment of appropriately selected spinal epidural AVFs. Cord compression from an enlarging venous varix can be treated concurrently with endovascular occlusion of an associated spinal epidural AVF.

An In Vitro Cynomolgus Vascular Surrogate System for Preclinical Drug Assessment and Human Translation.

OBJECTIVES: The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. APPROACH AND RESULTS: We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. CONCLUSIONS: The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.

Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of monocyte chemoattractant protein-1.

OBJECTIVE: Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. APPROACH AND RESULTS: We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet-fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. CONCLUSIONS: These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1-dependent mechanisms.

Human coronary artery perivascular adipocytes overexpress genes responsible for regulating vascular morphology, inflammation, and hemostasis.

Inflammatory cross talk between perivascular adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We previously reported that human perivascular (PV) adipocytes exhibit a proinflammatory phenotype and less adipogenic differentiation than do subcutaneous (SQ) adipocytes. To gain a global view of the genomic basis of biologic differences between PV and SQ adipocytes, we performed genome-wide expression analyses to identify differentially expressed genes between adipocytes derived from human SQ vs. PV adipose tissues. Although >90% of well-expressed genes were similarly regulated, we identified a signature of 307 differentially expressed genes that were highly enriched for functions associated with the regulation of angiogenesis, vascular morphology, inflammation, and blood clotting. Of the 156 PV upregulated genes, 59 associate with angiogenesis, vascular biology, or inflammation, noteworthy of which include TNFRSF11B (osteoprotegerin), PLAT, TGFB1, THBS2, HIF1A, GATA6, and SERPINE1. Of 166 PV downregulated genes, 21 associated with vascular biology and inflammation, including ANGPT1, ANGPTL1, and VEGFC. Consistent with the emergent hypothesis that PV adipocytes differentially regulate angiogenesis and inflammation, cell culture-derived adipocyte-conditioned media from PV adipocytes strongly enhanced endothelial cell tubulogenesis and monocyte migration compared with media from SQ adipocytes. These findings demonstrate that PV adipocytes have the potential to significantly modulate vascular inflammatory crosstalk in the setting of atherosclerosis by their ability to signal to both endothelial and inflammatory cells.

Histone deacetylase 9 is a negative regulator of adipogenic differentiation.

Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBPα gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBPα promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBPα expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.

Crosstalk between perivascular adipose tissue and blood vessels.

Crosstalk between cells in the blood vessel wall is vital to normal vascular function and is perturbed in diseases such as atherosclerosis and hypertension. Perivascular adipocytes reside at the adventitial border of blood vessels but until recently were virtually ignored in studies of vascular function. However, perivascular adipocytes have been demonstrated to be powerful endocrine cells capable of responding to metabolic cues and transducing signals to adjacent blood vessels. Accordingly, crosstalk between perivascular adipose tissue (PVAT) and blood vessels is now being intensely examined. Emerging evidence suggests that PVAT regulates vascular function through numerous mechanisms, but evidence to date suggests modulation of three key aspects that are the focus of this review: inflammation, vasoreactivity, and smooth muscle cell proliferation.

Estrogen receptor-alpha expression in the mammary epithelium is required for ductal and alveolar morphogenesis in mice.

The estrogen receptor-alpha (ERalpha) is a critical transcription factor that regulates epithelial cell proliferation and ductal morphogenesis during postnatal mammary gland development. Tissue recombination and transplantation studies using the first generation of ERalpha knockout (ERKO) mice suggested that this steroid hormone receptor is required in the mammary stroma that subsequently exerts its effect on the epithelium through additional paracrine signaling events. A more detailed analysis revealed that ERKO mice produce a truncated ERalpha protein with detectable transactivation activity, and it is likely that this functional ERalpha variant has masked the biological significance of this steroid receptor in the mammary epithelium. In this article, we describe the generation a Cre-lox-based conditional knockout of the ERalpha gene to study the biological function of this steroid receptor in the epithelial compartment at defined stages of mammary gland development. The mouse mammary tumor virus (MMTV)-Cre-mediated, epithelial-specific ablation of exon 3 of the ERalpha gene in virgin mice severely impaired ductal elongation and side branching. The conditional knockout resulted in ablation of the ERalpha protein, and the progesterone receptor (PR), whose expression is under the control of ERalpha, was largely absent. The whey acidic protein (WAP)-Cre-mediated deletion of ERalpha during successive gestation cycles resulted in a loss of ductal side-branching and lobuloalveolar structures, ductal dilation, and decreased proliferation of alveolar progenitors. These abnormalities compromised milk production and led to malnourishment of the offspring by the second lactation. These observations suggest that ERalpha expression in the mammary epithelium is essential for normal ductal morphogenesis during puberty and alveologenesis during pregnancy and lactation.

Bcl-2/adenovirus E1B 19 kDa interacting protein-3 knockdown enables growth of breast cancer metastases in the lung, liver, and bone.

The mouse breast cancer cell lines 4T1, 4T07, and 67NR are highly tumorigenic but vary in metastatic potential: 4T1 widely disseminates, resulting in secondary tumors in the lung, liver, bone, and brain; 4T07 spreads to the lung and liver but is unable to establish metastatic nodules; 67NR is unable to metastasize. The Bcl-2/adenovirus E1B 19 kDa interacting protein-3 (Bnip-3) was recently shown to be absent after hypoxia in pancreatic cancer cell lines whereas its overexpression restored hypoxia-induced cell death. We found that Bnip-3 expression increased after 6 hours of hypoxia in all cell lines tested but was highest in the nonmetastatic 67NR cells and lowest in the highly metastatic 4T1 cells. Hypoxia-induced expression of Bnip-3 in the disseminating but nonmetastatic 4T07 cells was intermediate compared with 4T1 and 67NR cells. Cleaved caspase-3, a key downstream effector of cell death, increased after 6 hours of hypoxia in the 67NR and 4T07 cells by 1.9- and 2.5-fold, respectively. Conversely, cleaved caspase-3 decreased by 45% in the highly metastatic 4T1 cells after hypoxia. Small interfering RNA oligonucleotides targeting endogenous Bnip-3 blocked cell death and increased clonigenic survival after hypoxic challenge in vitro and increased primary tumor size and enabled metastasis to the lung, liver, and sternum of mice inoculated with 4T07 cells in vivo. These data inversely correlate the hypoxia-induced expression of the cell death protein Bnip-3 to metastatic potential and suggest that loss of Bnip-3 expression is critical for malignant and metastatic evasion of hypoxia-induced cell death.

Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS.

Neurotrophin treatment has so far failed to prolong the survival of individuals affected with amyotrophic lateral sclerosis (ALS), an incurable motoneuron degenerative disorder. Here we show that intracerebroventricular (i.c.v.) delivery of recombinant vascular endothelial growth factor (Vegf) in a SOD1(G93A) rat model of ALS delays onset of paralysis by 17 d, improves motor performance and prolongs survival by 22 d, representing the largest effects in animal models of ALS achieved by protein delivery. By protecting cervical motoneurons, i.c.v. delivery of Vegf is particularly effective in rats with the most severe form of ALS with forelimb onset. Vegf has direct neuroprotective effects on motoneurons in vivo, because neuronal expression of a transgene expressing the Vegf receptor prolongs the survival of SOD1(G93A) mice. On i.c.v. delivery, Vegf is anterogradely transported and preserves neuromuscular junctions in SOD1(G93A) rats. Our findings in preclinical rodent models of ALS may have implications for treatment of neurodegenerative disease in general.

Critical role of platelet P-selectin in the response to arterial injury in apolipoprotein-E-deficient mice.

OBJECTIVE: Mice deficient in apolipoprotein-E (apoE-/-) experience severe hypercholesterolemia that is exacerbated by a high-fat Western-type diet and atherosclerotic lesions spontaneously develop. In addition, we have reported that deficiency of P-selectin dramatically protects against neointimal lesion formation after arterial injury in apoE-/- mice. To define the mechanism, bone marrow transplantation (BMT) after lethal irradiation was used to generate apoE-/- chimeric mice deficient in platelet, but not endothelial, P-selectin. METHODS AND RESULTS: Mice underwent vascular injury and were euthanized 4 weeks later. Absence of platelet P-selectin (pPS) expression in apoE-/- mice after BMT was confirmed by flow cytometry and Western blot analysis. Lack of pPS in apoE-/- mice resulted in a 62% reduction in neointimal area (45 000+/-27 000 versus 17 000+/-13 000 microm2, P<0.000001) and a 30% reduction (P<0.02) in macrophage infiltration, compared with control apoE-/- BMT. Absence of pPS was also associated with a reduction in plaque neovascularization as compared with pPS-competent controls (0/8 versus 3/8, P<0.05). CONCLUSIONS: Lack of pPS significantly attenuates macrophage recruitment and neointimal lesion formation, indicating that pPS on platelets lining the vessel wall plays a critical role in inflammation after wire-withdrawal injury of the carotid artery in apoE-/- mice.

Single injection of P-selectin or P-selectin glycoprotein ligand-1 monoclonal antibody blocks neointima formation after arterial injury in apolipoprotein E-deficient mice.

BACKGROUND: Emerging data suggest that P-selectin, by controlling adhesion of white blood cells, may be important in limiting the response to vascular injury. METHODS AND RESULTS: We tested the hypothesis that transient inhibition of P-selectin with either anti-P-selectin monoclonal antibody (mAb) or anti-P-selectin glycoprotein ligand-1 (PSGL-1) mAb would reduce neointima formation in the setting of carotid denudation injury in atherosclerosis-prone apolipoprotein E-/- mice. Neointima formation at 28 days was reduced significantly, by 50% or 80%, by a single injection on the day of injury of 100 or 200 microg P-selectin mAb RB 40.34 and by 55% by a single injection of 100 microg PSGL-1 mAb 4RA10 (P< or =0.005). In addition, there was a significant reduction in neointimal macrophage content. CONCLUSIONS: These findings demonstrate that transient P-selectin or PSGL-1 blockade at the time of arterial injury significantly limits plaque macrophage content and neointima formation in a dose-dependent manner after carotid denudation injury in apolipoprotein E-/- mice.

Deposition of platelet RANTES triggering monocyte recruitment requires P-selectin and is involved in neointima formation after arterial injury.

BACKGROUND: Chemokines expressed on atherosclerotic endothelium or deposited by activated platelets have been implicated in monocyte recruitment during atherogenesis and restenosis. Although the involvement of P-selectin in these processes is evident from studies in knockout mice, it has not been elucidated whether delivery of platelet chemokines requires P-selectin, thus serving as a P-selectin-dependent effector function. METHODS AND RESULTS: Using immunofluorescence and laminar flow assays, we found that the deposition of the platelet-derived chemokine RANTES and monocyte arrest subsequently triggered by RANTES immobilized on inflamed endothelium are more efficient after preperfusion than after static preincubation of platelets and appear to depend on interactions of platelet but not endothelial P-selectin. This was revealed by the effects of P-selectin antibodies and comparison of P-selectin-deficient and wild-type platelets. Immunohistochemistry detected a substantial luminal expression of RANTES on neointimal lesions in wire-injured carotid arteries of apolipoprotein E (apoE)-deficient mice but not of mice with a combined deficiency in apoE and P-selectin (or platelet P-selectin). As assessed by histomorphometry, treatment of apoE-deficient mice with the RANTES receptor antagonist Met-RANTES markedly reduced neointimal plaque area and macrophage infiltration. CONCLUSIONS: Our data suggest that RANTES deposition and subsequent monocyte arrest are promoted by platelet P-selectin and involved in wire-induced intimal hyperplasia, and that blocking RANTES receptors attenuates neointima formation and macrophage infiltration. This mechanism represents an important component explaining the protection against neointimal growth in P-selectin-deficient mice and may represent a novel approach to the treatment of restenosis or atherosclerosis by the administration of chemokine receptor antagonists.