Zia A. Khan

Engineering Robust and Functional Vascular Networks In Vivo With Human Adult and Cord Blood–Derived Progenitor Cells

The success of therapeutic vascularization and tissue engineering will rely on our ability to create vascular networks using human cells that can be obtained readily, can be expanded safely ex vivo, and can produce robust vasculogenic activity in vivo. Here we describe the formation of functional microvascular beds in immunodeficient mice by coimplantation of human endothelial and mesenchymal progenitor cells isolated from blood and bone marrow. Evaluation of implants after 1 week revealed an extensive network of human blood vessels containing erythrocytes, indicating the rapid formation of functional anastomoses within the host vasculature. The implanted endothelial progenitor cells were restricted to the luminal aspect of the vessels; mesenchymal progenitor cells were adjacent to lumens, confirming their role as perivascular cells. Importantly, the engineered vascular networks remained patent at 4 weeks in vivo. This rapid formation of long-lasting microvascular networks by postnatal progenitor cells obtained from noninvasive sources constitutes an important step forward in the development of clinical strategies for tissue vascularization.

Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice

Infantile hemangioma is a benign endothelial tumor composed of disorganized blood vessels. It exhibits a unique life cycle of rapid postnatal growth followed by slow regression to a fibrofatty residuum. Here, we have reported the isolation of multipotential stem cells from hemangioma tissue that give rise to hemangioma-like lesions in immunodeficient mice. Cells were isolated based on expression of the stem cell marker CD133 and expanded from single cells as clonal populations. The CD133-selected cells generated human blood vessels 7 days after implantation in immunodeficient mice. Cell retrieval experiments showed the cells could again form vessels when transplanted into secondary recipients. The human vessels expressed GLUT-1 and merosin, immunodiagnostic markers for infantile hemangioma. Two months after implantation, the number of blood vessels diminished and human adipocytes became evident. Lentiviral expression of GFP was used to confirm that the hemangioma-derived cells formed the blood vessels and adipocytes in the immunodeficient mice. Thus, when transplanted into immunodeficient mice, hemangioma-derived cells recapitulated the unique evolution of infantile hemangioma--the formation of blood vessels followed by involution to fatty tissue. In summary, this study identifies a stem cell as the cellular origin of infantile hemangioma and describes for what we believe is the first time an animal model for this common tumor of infancy.

Curcumin prevents diabetes-associated abnormalities in the kidneys by inhibiting p300 and nuclear factor-κB

OBJECTIVE Diabetic nephropathy is a debilitating disease that leads to end-stage renal failure in the Western world. Hyperglycemia is the initiating factor in several chronic diabetic complications which mediates increased oxidative stress and eventually the increased production of vasoactive factors and extracellular matrix proteins. We hypothesized that curcumin, a potent antioxidant, might be beneficial in preventing the development of diabetic nephropathy because this compound has been shown to inhibit p300, a histone acetyltransferase that plays a role in regulating gene expression through its interaction with the transcription factor nuclear factor-kappaB. METHODS To test this hypothesis, male Sprague-Dawley rats were injected with streptozotocin to induce diabetes. These animals were subsequently treated with curcumin for a period of 1 mo. RESULTS Real-time reverse transcriptase polymerase chain reaction analyses showed that diabetes-induced upregulation of vasoactive factors (endothelial nitric oxide synthase and endothelin-1), transforming growth factor-beta1 and extracellular matrix proteins (fibronectin and extradomain-B-containing fibronectin) in the kidneys. These changes were associated with increased oxidative stress, mesangial expansion, and p300 and nuclear factor-kappaB activity that were prevented with curcumin treatment. CONCLUSION These beneficial effects of curcumin were mediated through the inhibition of p300 and nuclear factor-kappaB.

Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-β2

In situ analysis of fetal semilunar valve leaflets has revealed cells coexpressing endothelial and mesenchymal markers along the endothelium, with diminished frequency seen in adult valves. To determine whether such cells are progenitor cells, we isolated clonal populations from human pulmonary valves. The clones expressed endothelial markers but showed potential to further differentiate into endothelium in response to vascular endothelial growth factor (VEGF)-A. When exposed to transforming growth factor (TGF)-&bgr;2, individual clones adopted a mesenchymal phenotype to varying degrees and expressed markers of endothelial to mesenchymal transformation (EMT). Both VEGF- and TGF&bgr;2–induced phenotypic changes were partially reversible, indicating the plasticity of these cells. When challenged with VEGF or TGF&bgr;2, a hierarchy of endothelial/mesenchymal potential could be seen among the clonal populations: cells initially closer to an endothelial phenotype showed a strong response to TGF&bgr;2 that could be inhibited by VEGF, whereas cells closer to a mesenchymal phenotype responded to TGF&bgr;2 but were resistant to endothelial-inducing effects of VEGF. These findings suggest the presence of bipotential valve progenitor cells with ability to differentiate into either endothelial or interstitial cells of the valve leaflet. Understanding the differentiation potential and function of these cells may be important for understanding heart valve disease and may also be applied to current paradigms for creating tissue-engineered heart valves.

Calcification of multipotent prostate tumor endothelium.

Solid tumors require new blood vessels for growth and metastasis, yet the biology of tumor-specific endothelial cells is poorly understood. We have isolated tumor endothelial cells from mice that spontaneously develop prostate tumors. Clonal populations of tumor endothelial cells expressed hematopoietic and mesenchymal stem cell markers and differentiated to form cartilage- and bone-like tissues. Chondrogenic differentiation was accompanied by an upregulation of cartilage-specific col2a1 and sox9, whereas osteocalcin and the metastasis marker osteopontin were upregulated during osteogenic differentiation. In human and mouse prostate tumors, ectopic vascular calcification was predominately luminal and colocalized with the endothelial marker CD31. Thus, prostate tumor endothelial cells are atypically multipotent and can undergo a mesenchymal-like transition.

Diabetes-Induced Extracellular Matrix Protein Expression Is Mediated by Transcription Coactivator p300

Increased fibronectin expression is a key feature of diabetic angiopathy. We have previously shown that nuclear factor-κB (NF-κB) mediates fibronectin expression in endothelial cells and in organs affected by diabetes complications. p300, a transcription coactivator, may regulate NF-κB activity via poly(ADP-ribose) polymerase (PARP) activation. Hence, we examined the role of p300 in fibronectin expression in diabetes. High glucose induced fibronectin expression in the endothelial cells, which was associated with increased p300, PARP activity, and NF-κB activation. This p300 alteration is mediated by mitogen-activated protein kinase and protein kinase C and B. We then used p300 small interfering RNA (siRNA) and showed decreased fibronectin and PARP expression, as well as NF-κB activation, in the endothelial cells. Examination of the heart tissues of streptozotocin-induced diabetic mice revealed increased fibronectin and p300 mRNA. Intravenous injection of p300 siRNA resulted in decreased p300 levels and normalized fibronectin expression in the heart. We further investigated retinal tissues from streptozotocin-induced diabetic rats treated with intravitreal p300 siRNA injection. Similar to the heart, p300 siRNA inhibited fibronectin expression in the retina of the diabetic animals. These results indicate that transcriptional coactivator p300 may regulate fibronectin expression via PARP and NF-κB activation in diabetes.

Cellular Signaling and Potential New Treatment Targets in Diabetic Retinopathy

Dysfunction and death of microvascular cells and imbalance between the production and the degradation of extracellular matrix (ECM) proteins are a characteristic feature of diabetic retinopathy (DR). Glucose-induced biochemical alterations in the vascular endothelial cells may activate a cascade of signaling pathways leading to increased production of ECM proteins and cellular dysfunction/death. Chronic diabetes leads to the activation of a number of signaling proteins including protein kinase C, protein kinase B, and mitogen-activated protein kinases. These signaling cascades are activated in response to hyperglycemia-induced oxidative stress, polyol pathway, and advanced glycation end product formation among others. The aberrant signaling pathways ultimately lead to activation of transcription factors such as nuclear factor-κB and activating protein-1. The activity of these transcription factors is also regulated by epigenetic mechanisms through transcriptional coactivator p300. These complex signaling pathways may be involved in glucose-induced alterations of endothelial cell phenotype leading to the production of increased ECM proteins and vasoactive effector molecules causing functional and structural changes in the microvasculature. Understanding of such mechanistic pathways will help to develop future adjuvant therapies for diabetic retinopathy.

EDB fibronectin and angiogenesis -- a novel mechanistic pathway

Extra domain-B containing fibronectin (EDB+ FN), a recently proposed marker of angiogenesis, has been shown to be expressed in a number of human cancers and in ocular neovascularization in patients with proliferative diabetic retinopathy. To gain molecular understanding of the functional significance of EDB+ FN, we have investigated possible regulatory mechanisms of induction and its role in endothelial cell proliferation and angiogenesis. Human vascular endothelial cells were cultured in high levels of glucose, and fibrogenic growth factors, transforming growth factor-β1 (TGF-β1) and endothelin-1 (ET-1). Our results show that high glucose levels, TGF-β1, and ET-1 upregulated EDB+ FN expression. Treatment of cells exposed to high glucose with TGF-β1 neutralizing antibody and ET receptor antagonist prevented high glucose-induced EDB+ FN expression. In order to elucidate the functional significance of EDB+ FN upregulation, cells were subjected to in vitro proliferation and angiogenesis assays following EDB peptide treatment and specific EDB+ FN gene silencing. Our results show that exposure of cells to EDB peptide increased vascular endothelial growth factor (VEGF) expression, endothelial proliferation, and tube formation. Furthermore, specific EDB+ FN gene silencing prevented both basal and high glucose-induced VEGF expression and reduced the proliferative capacity of endothelial cells. In conclusion, these results indicate that EDB+ FN is involved in endothelial cell proliferation and vascular morphogenesis, findings which may provide novel avenues for the development of anti-angiogenic therapies.

Curcumin protects hearts from FFA-induced injury by activating Nrf2 and inactivating NF-κB both in vitro and in vivo.

Obesity and increased free fatty acid (FFA) level are tightly linked, leading to the development of cardiovascular disorders. Curcumin is a natural product from Curcuma longa with multiple bioactivities and is known to have cardioprotective effects in several cellular and animal models. The current study was designed to evaluate the cardioprotective effects of curcumin and demonstrate the underlying mechanism in FFA-induced cardiac injury. Using cell culture studies and high fat in vivo model, we explored the mechanistic basis of anti-inflammatory and antioxidant activities of curcumin. We observed that palmitate (PA) treatment in cardiac derived H9C2 cells induced a marked increase in reactive oxygen species, inflammation, apoptosis and hypertrophy. All of these changes were effectively suppressed by curcumin treatment. In addition, oral administration of curcumin at 50mg/kg completely suppressed high fat diet-induced oxidative stress, inflammation, apoptosis, fibrosis, hypertrophy and tissue remodeling in mice. The beneficial actions of curcumin are closely associated with its ability to increase Nrf2 expression and inhibit NF-κB activation. Thus, both in vitro and in vivo studies showed a promising role of curcumin as a cardioprotective agent against palmitate and high fat diet mediated cardiac dysfunction. We indicated the regulatory roles of Nrf2 and NF-κB in obesity-induced heart injury, and suggested that they may be important therapeutic targets in the treatment of obesity-related disorders.

Extracellular signal-regulated kinase (ERK) in glucose-induced and endothelin-mediated fibronectin synthesis.

Increased extracellular matrix protein deposition and basement membrane thickening are important features of diabetic angiopathy. One key matrix protein that has been shown to be instrumental in basement membrane thickening is fibronectin (FN). We have previously demonstrated that glucose-induced increased expression of endothelin-1 (ET-1), may in part, be responsible for increased FN expression via nuclear factor-κB (NF-κB) and activating protein (AP-1) activation. The present study was aimed at elucidating the mechanism of ET-1 with respect to mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway activation and glucose-induced FN upregulation. Human endothelial cells were exposed to either low (5 mM) or high (25 mM) glucose levels. Cells in low glucose were also treated with ET-1 peptide (5 nM). In addition, we treated cells exposed to high glucose levels with specific MAPK/ERK inhibitor PD098059 (50 μM), dual ET-receptor antagonist, bosentan (10 μM), and PKC blocker, chelerythrine (1 μM). Following incubation period, RNA and total proteins were extracted for RT-PCR for FN and immunoblot analysis of MAPK/ERK activation. Confocal microscopy was performed for analysis of FN protein and nuclear localization of activated Elk. Electrophoretic mobility shift assay was carried out to detect NF-κB and AP-1 activation. Our data demonstrates that high glucose-induced upregulation of FN messenger RNA and protein levels occur via activation of MAPK/ERK pathway, which was prevented by treatment of cells with bosentan, PD098059 and PKC blocker chelerythrine. Confocal microscopy demonstrated nuclear localization of phospho-Elk protein. Glucose-induced FN expression was also associated with protein kinase C, NF-κB, and AP-1 activation. These results suggested that glucose-induced, ET- and PKC-dependent, upregulation of FN is, in part, mediated via MAPK/ERK activation.