Biomedical Engineer, Veterans Affairs Palo Alto Health Care System (2012 - Present)
Assistant Professor, Cardiothoracic Surgery (2013 - Present)
Instructor, School of Medicine (2010 - 2012)
Honors & Awards
Robert W Hobson II MD Early Career Investigator Award, American Heart Association Council on Peripheral Vascular Disease (2012)
Jay D. Coffman Young Investigator Award, First Place in Basic Science, Society for Vascular Medicine (2011)
K99/R00 Pathways to Independence, NIH (09/01/10-Present)
National Scientist Development Grant, American Heart Association (06/01/10-08/31/10)
NRSA Postdoctoral Fellowship, NIH (2009-2010)
Postdoctoral Fellowship, American Heart Association (2008-2009)
Doctor of Philosophy, University of CA Berkeley, Bioengineering (2006)
Master of Science, University of CA Berkeley, Bioengineering (2005)
Bachelor of Science, MIT, Chemical Engineering (2002)
Current Research and Scholarly Interests
Dr. Huangs laboratory aims to understand the chemical and mechanical interactions between extracellular matrix (ECM) proteins and pluripotent stem cells that regulate vascular and myogenic differentiation. The fundamental insights of cell-matrix interactions are applied towards stem cell-based therapies with respect to improving cell survival and regenerative capacity, as well as engineered vascularized tissues for therapeutic implantation. Current projects focus on the role of naturally-derived ECMs to enhance endothelial differentiation of induced pluripotent stem cells on two-dimensional ECM microarrays of varying substrate rigidity. The knowledge gained from understanding cell-ECM interactions are applied towards engineering prevascularized skeletal or cardiac muscle constructs using nanotopographical cues derived from nanofibrillar ECMs.
Microvascular Endothelial Cells Migrate Upstream and Align Against the Shear Stress Field Created by Impinging Flow
2014; 106 (2): 366-374
At present, little is known about how endothelial cells respond to spatial variations in fluid shear stress such as those that occur locally during embryonic development, at heart valve leaflets, and at sites of aneurysm formation. We built an impinging flow device that exposes endothelial cells to gradients of shear stress. Using this device, we investigated the response of microvascular endothelial cells to shear-stress gradients that ranged from 0 to a peak shear stress of 9-210 dyn/cm(2). We observe that at high confluency, these cells migrate against the direction of fluid flow and concentrate in the region of maximum wall shear stress, whereas low-density microvascular endothelial cells that lack cell-cell contacts migrate in the flow direction. In addition, the cells align parallel to the flow at low wall shear stresses but orient perpendicularly to the flow direction above a critical threshold in local wall shear stress. Our observations suggest that endothelial cells are exquisitely sensitive to both magnitude and spatial gradients in wall shear stress. The impinging flow device provides a, to our knowledge, novel means to study endothelial cell migration and polarization in response to gradients in physical forces such as wall shear stress.
View details for DOI 10.1016/j.bpj.2013.11.4502
View details for Web of Science ID 000330132500005
View details for PubMedID 24461011
Characterization of a Fluorescent Probe for Imaging Nitric Oxide
JOURNAL OF VASCULAR RESEARCH
2014; 51 (1): 68-79
Nitric oxide (NO), a potent vasodilator and anti-atherogenic molecule, is synthesized in various cell types, including vascular endothelial cells (ECs). The biological importance of NO enforces the need to develop and characterize specific and sensitive probes. To date, several fluorophores, chromophores and colorimetric techniques have been developed to detect NO or its metabolites (NO(2) and NO(3)) in biological fluids, viable cells or cell lysates.Recently, a novel probe (NO(550)) has been developed and reported to detect NO in solutions and in primary astrocytes and neuronal cells with a fluorescence signal arising from a nonfluorescent background.Here, we report further characterization of this probe by optimizing conditions for the detection and imaging of NO products in primary vascular ECs, fibroblasts, and embryonic stem cell- and induced pluripotent stem cell-derived ECs in the absence and presence of pharmacological agents that modulate NO levels. In addition, we studied the stability of this probe in cells over time and evaluated its compartmentalization in reference to organelle-labeling dyes. Finally, we synthesized an inherently fluorescent diazo ring compound (AZO(550)) that is expected to form when the nonfluorescent NO(550) reacts with cellular NO, and compared its cellular distribution with that of NO(550).NO(550) is a promising agent for imaging NO at baseline and in response to pharmacological agents that modulate its levels.
View details for DOI 10.1159/000356445
View details for Web of Science ID 000329771700007
View details for PubMedID 24335468
Effects of Dimethylarginine Dimethylaminohydrolase-1 Overexpression on the Response of the Pulmonary Vasculature to Hypoxia
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY
2013; 49 (3): 491-500
Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1(tg)) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3',5'-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1(tg) mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1(tg). The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1(tg) mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.
View details for DOI 10.1165/rcmb.2012-0330OC
View details for Web of Science ID 000324195400018
View details for PubMedID 23642043
Conversion of Human Fibroblasts to Functional Endothelial Cells by Defined Factors
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2013; 33 (6): 1366-?
Transdifferentiation of fibroblasts to endothelial cells (ECs) may provide a novel therapeutic avenue for diseases, including ischemia and fibrosis. Here, we demonstrate that human fibroblasts can be transdifferentiated into functional ECs by using only 2 factors, Oct4 and Klf4, under inductive signaling conditions.To determine whether human fibroblasts could be converted into ECs by transient expression of pluripotency factors, human neonatal fibroblasts were transduced with lentiviruses encoding Oct4 and Klf4 in the presence of soluble factors that promote the induction of an endothelial program. After 28 days, clusters of induced endothelial (iEnd) cells seemed and were isolated for further propagation and subsequent characterization. The iEnd cells resembled primary human ECs in their transcriptional signature by expressing endothelial phenotypic markers, such as CD31, vascular endothelial-cadherin, and von Willebrand Factor. Furthermore, the iEnd cells could incorporate acetylated low-density lipoprotein and form vascular structures in vitro and in vivo. When injected into the ischemic limb of mice, the iEnd cells engrafted, increased capillary density, and enhanced tissue perfusion. During the transdifferentiation process, the endogenous pluripotency network was not activated, suggesting that this process bypassed a pluripotent intermediate step.Pluripotent factor-induced transdifferentiation can be successfully applied for generating functional autologous ECs for therapeutic applications.
View details for DOI 10.1161/ATVBAHA.112.301167
View details for Web of Science ID 000319119500038
View details for PubMedID 23520160
The modulation of endothelial cell morphology, function, and survival using anisotropic nanofibrillar collagen scaffolds
2013; 34 (16): 4038-4047
Endothelial cells (ECs) are aligned longitudinally under laminar flow, whereas they are polygonal and poorly aligned in regions of disturbed flow. The unaligned ECs in disturbed flow fields manifest altered function and reduced survival that promote lesion formation. We demonstrate that the alignment of the ECs may directly influence their biology, independent of fluid flow. We developed aligned nanofibrillar collagen scaffolds that mimic the structure of collagen bundles in blood vessels, and examined the effects of these materials on EC alignment, function, and in vivo survival. ECs cultured on 30-nm diameter aligned fibrils re-organized their F-actin along the nanofibril direction, and were 50% less adhesive for monocytes than the ECs grown on randomly oriented fibrils. After EC transplantation into both subcutaneous tissue and the ischemic hindlimb, EC viability was enhanced when ECs were cultured and implanted on aligned nanofibrillar scaffolds, in contrast to non-patterned scaffolds. ECs derived from human induced pluripotent stem cells and cultured on aligned scaffolds also persisted for over 28 days, as assessed by bioluminescence imaging, when implanted in ischemic tissue. By contrast, ECs implanted on scaffolds without nanopatterning generated no detectable bioluminescent signal by day 4 in either normal or ischemic tissues. We demonstrate that 30-nm aligned nanofibrillar collagen scaffolds guide cellular organization, modulate endothelial inflammatory response, and enhance cell survival after implantation in normal and ischemic tissues.
View details for DOI 10.1016/j.biomaterials.2013.02.036
View details for Web of Science ID 000317322600011
View details for PubMedID 23480958
Spatial patterning of endothelium modulates cell morphology, adhesiveness and transcriptional signature
2013; 34 (12): 2928-2937
Microscale and nanoscale structures can spatially pattern endothelial cells (ECs) into parallel-aligned organization, mimicking their cellular alignment in blood vessels exposed to laminar shear stress. However, the effects of spatial patterning on the function and global transcriptome of ECs are incompletely characterized. We used both parallel-aligned micropatterned and nanopatterned biomaterials to evaluate the effects of spatial patterning on the phenotype of ECs, based on gene expression profiling, functional characterization of monocyte adhesion, and quantification of cellular morphology. We demonstrate that both micropatterned and aligned nanofibrillar biomaterials could effectively guide EC organization along the direction of the micropatterned channels or nanofibrils, respectively. The ability of ECs to sense spatial patterning cues were abrogated in the presence of cytoskeletal disruption agents. Moreover, both micropatterned and aligned nanofibrillar substrates promoted an athero-resistant EC phenotype by reducing endothelial adhesiveness for monocytes and platelets, as well as by downregulating the expression of adhesion proteins and chemokines. We further found that micropatterned ECs have a transcriptional signature that is unique from non-patterned ECs, as well as from ECs aligned by shear stress. These findings highlight the importance of spatial patterning cues in guiding EC organization and function, which may have clinical relevance in the development of vascular grafts that promote patency.
View details for DOI 10.1016/j.biomaterials.2013.01.017
View details for Web of Science ID 000316038900008
View details for PubMedID 23357369
Chemotaxis of human induced pluripotent stem cell-derived endothelial cells
AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH
2013; 5 (5): 510-U96
This study examined the homing capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and their response to chemotactic gradients of stromal derived factor-1α (SDF). We have previously shown that EC derived from murine pluripotent stem cells can home to the ischemic hindlimb of the mouse. In the current study, we were interested to understand if ECs derived from human induced pluripotent stem cells are capable of homing. The homing capacity of iPSC-ECs was assessed after systemic delivery into immunodeficient mice with unilateral hindlimb ischemia. Furthermore, the iPSC-ECs were evaluated for their expression of CXCR4 and their ability to respond to SDF chemotactic gradients in vitro. Upon systemic delivery, the iPSC-ECs transiently localized to the lungs but did not home to the ischemic limb over the course of 14 days. To understand the mechanism of the lack of homing, the expression levels of the homing receptor, CXCR4, was examined at the transcriptional and protein levels. Furthermore, their ability to migrate in response to chemokines was assessed using microfluidic and scratch assays. Unlike ECs derived from syngeneic mouse pluripotent stem cells, human iPSC-ECs do not home to the ischemic mouse hindlimb. This lack of functional homing may represent an impairment of interspecies cellular communication or a difference in the differentiation state of the human iPSC-ECs. These results may have important implications in therapeutic delivery of iPSC-ECs.
View details for Web of Science ID 000323539100004
View details for PubMedID 23977410
Human induced pluripotent stem cell-derived endothelial cells exhibit functional heterogeneity.
American journal of translational research
2013; 5 (1): 21-35
Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are promising for treatment of vascular diseases. However, hiPSC-ECs purified based on CD31 expression are comprised of arterial, venous, and lymphatic subtypes. It is unclear whether hiPSC-ECs are heterogeneous in nature, and whether there may be functional benefits of enriching for specific subtypes. Therefore, we sought to characterize the hiPSC-ECs and enrich for each subtype, and demonstrate whether such enrichment would have functional significance. The hiPSC-ECs were generated from differentiation of hiPSCs using vascular endothelial growth factor (VEGF)-A and bone morphogenetic protein-4. The hiPSC-ECs were purified based on positive expression of CD31. Subsequently, we sought to enrich for each subtype. Arterial hiPSC-ECs were induced using higher concentrations of VEGF-A and 8-bromoadenosine-3':5'-cyclic monophosphate in the media, whereas lower concentrations of VEGF-A favored venous subtype. VEGF-C and angiopoietin-1 promoted the expression of lymphatic phenotype. Upon FACS purification based on CD31+ expression, the hiPSC-EC population was observed to display typical endothelial surface markers and functions. However, the hiPSC-EC population was heterogeneous in that they displayed arterial, venous, and to a lesser degree, lymphatic lineage markers. Upon comparing vascular formation in matrigel plugs in vivo, we observed that arterial enriched hiPSC-ECs formed a more extensive capillary network in this model, by comparison to a heterogeneous population of hiPSC-ECs. This study demonstrates that FACS purification of CD31+ hiPSC-ECs produces a diverse population of ECs. Refining the differentiation methods can enrich for subtype-specific hiPSC-ECs with functional benefits of enhancing neovascularization.
View details for PubMedID 23390563
Multifunctional in vivo vascular imaging using near-infrared II fluorescence
2012; 18 (12): 1841-?
In vivo real-time epifluorescence imaging of mouse hind limb vasculatures in the second near-infrared region (NIR-II) is performed using single-walled carbon nanotubes as fluorophores. Both high spatial (?30 ?m) and temporal (<200 ms per frame) resolution for small-vessel imaging are achieved at 1-3 mm deep in the hind limb owing to the beneficial NIR-II optical window that affords deep anatomical penetration and low scattering. This spatial resolution is unattainable by traditional NIR imaging (NIR-I) or microscopic computed tomography, and the temporal resolution far exceeds scanning microscopic imaging techniques. Arterial and venous vessels are unambiguously differentiated using a dynamic contrast-enhanced NIR-II imaging technique on the basis of their distinct hemodynamics. Further, the deep tissue penetration and high spatial and temporal resolution of NIR-II imaging allow for precise quantifications of blood velocity in both normal and ischemic femoral arteries, which are beyond the capabilities of ultrasonography at lower blood velocities.
View details for DOI 10.1038/nm.2995
View details for Web of Science ID 000311999800033
View details for PubMedID 23160236
Endothelial Cells Derived From Nuclear Reprogramming
2012; 111 (10): 1363-1375
The endothelium plays a pivotal role in vascular homeostasis, regulating the tone of the vascular wall, and its interaction with circulating blood elements. Alterations in endothelial functions facilitate the infiltration of inflammatory cells and permit vascular smooth muscle proliferation and platelet aggregation. Therefore, endothelial dysfunction is an early event in disease processes including atherosclerosis, and because of its critical role in vascular health, the endothelium is worthy of the intense focus it has received. However, there are limitations to studying human endothelial function in vivo, or human vascular segments ex vivo. Thus, methods for endothelial cell (EC) culture have been developed and refined. Recently, methods to derive ECs from pluripotent cells have extended the scientific range of human EC studies. Pluripotent stem cells may be generated, expanded, and then differentiated into ECs for in vitro studies. Constructs for molecular imaging can also be employed to facilitate tracking these cells in vivo. Furthermore, one can generate patient-specific ECs to study the effects of genetic or epigenetic alterations on endothelial behavior. Finally, there is the opportunity to apply these cells for vascular therapy. This review focuses on the generation of ECs from stem cells; their characterization by genetic, histological, and functional studies; and their translational applications.
View details for DOI 10.1161/CIRCRESAHA.111.247213
View details for Web of Science ID 000310501300017
View details for PubMedID 23104878
Aligned nanofibrillar collagen regulates endothelial organization and migration
2012; 7 (5): 649-661
Modulating endothelial cell (EC) morphology and motility, with the aim to influence their biology, might be beneficial for the treatment of vascular disease. We examined the effect of nanoscale matrix anisotropy on EC organization and migration for vascular tissue engineering applications.We developed a flow processing technique to generate anisotropic nanofibrillar collagen. Human ECs were cultured on aligned or on randomly oriented collagen, and their cellular alignment and cytoskeletal organization were characterized by immunofluorescence staining and time-lapse microscopy.ECs were elongated along the direction of aligned collagen nanofibrils and had organized focal adhesions. Cellular protrusion migrated with greater directionality and higher velocity along the anisotropic nanofibrils compared with cells on random nanofibrils. The flow technique can be adapted to fabricate vascular grafts that support the endothelial phenotype.Aligned nanofibrillar collagen regulates EC organization and migration, which can significantly contribute to the development of vascular grafts.
View details for DOI 10.2217/RME.12.48
View details for Web of Science ID 000308387900011
View details for PubMedID 22954436
Bioluminescence Imaging of Stem Cell-Based Therapeutics for Vascular Regeneration
2012; 2 (4): 346-354
Stem cell-based therapeutics show promise for treatment of vascular diseases. However, the survival of the cells after in vivo injection into diseased tissues remains a concern. In the advent of non-invasive optical imaging techniques such as bioluminescence imaging (BLI), cell localization and survival can be easily monitored over time. This approach has recently been applied towards monitoring stem cell treatments for vascular regeneration of the coronary or peripheral arteries. In this review, we will describe the application of BLI for tracking transplanted stem cells and associating their viability with therapeutic efficacy, in preclinical disease models of vascular disease.
View details for DOI 10.7150/thno.3694
View details for Web of Science ID 000304031200003
View details for PubMedID 22509198
Endothelial Cells Derived From Human iPSCS Increase Capillary Density and Improve Perfusion in a Mouse Model of Peripheral Arterial Disease
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2011; 31 (11): E72-U44
Stem cell therapy for angiogenesis and vascular regeneration has been investigated using adult or embryonic stem cells. In the present study, we investigated the potential of endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease.Endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with BMP-4 and vascular endothelial growth factor. The hiPSC-ECs exhibited endothelial characteristics by forming capillary-like structures in matrigel and incorporating acetylated-LDL. They stained positively for EC markers such as KDR, CD31, CD144, and eNOS. In vitro exposure of hiPSC-ECs to hypoxia resulted in increased expression of various angiogenic related cytokines and growth factors. hiPSC-ECs were stably transduced with a double fusion construct encoded by the ubiquitin promoter, firefly luciferase for bioluminescence imaging and green fluorescence protein for fluorescent detection. The hiPSC-ECs (5×10(5)) were delivered by intramuscular injection into the ischemic hindlimb of SCID mice at day 0 and again on day 7 after femoral artery ligation (n=8). Bioluminescence imaging showed that hiPSC-ECs survived in the ischemic limb for at least 2 weeks. In addition, laser Doppler imaging showed that the ratio of blood perfusion was increased by hiPSC-EC treatment by comparison to the saline-treated group (0.58±0.12 versus 0.44±0.04; P=0.005). The total number of capillaries in the ischemic limb of mice receiving hiPSC-EC injections was greater than those in the saline-treated group (1284±155 versus 797±206 capillaries/mm(2)) (P<0.002).This study is a first step toward development of a regenerative strategy for peripheral arterial disease based on the use of ECs derived from hiPSCs.
View details for DOI 10.1161/ATVBAHA.111.230938
View details for Web of Science ID 000296605400001
View details for PubMedID 21836062
Regulation of the Matrix Microenvironment for Stem Cell Engineering and Regenerative Medicine
ANNALS OF BIOMEDICAL ENGINEERING
2011; 39 (4): 1201-1214
The extracellular matrix (ECM) microenvironment consists of structural and functional molecules. The ECM relays both biochemical and biophysical cues to and from the cells to modulate cell behavior and function. The biophysical cues can be engineered and applied to cells by means of spatial patterning, matrix rigidity, and matrix actuation. Tissue engineering strategies that utilize ECMs to direct stem cell organization and lineage specification show tremendous potential. This review describes the technologies for modulating ECM spatial patterning, matrix rigidity, chemical composition, and matrix actuation. The role of ECMs in vascular tissue engineering is then discussed as a model of tissue engineering and regenerative medicine.
View details for DOI 10.1007/s10439-011-0297-2
View details for Web of Science ID 000289103500006
View details for PubMedID 21424849
Proteomic identification of biomarkers of vascular injury.
American journal of translational research
2011; 3 (2): 139-148
Predictive biomarkers may be beneficial for detecting, diagnosing, and assessing the risk of restenosis and vascular injury. We utilized proteomic profiling to identify protein markers in the blood following vascular injury, and corroborated the differential protein expression with immunological approaches. Rats underwent carotid artery injury, and plasma was collected after 2 or 5 weeks. Proteomic profiling was carried out by two-dimensional differential in-gel electrophoresis. The differentially expressed plasma proteins were identified by mass spectroscopy and confirmed by immunoblotting. Proteomic profiling by two-dimensional differential in-gel electrophoresis and mass spectroscopy revealed plasma proteins that were differentially expressed at 2 weeks after injury. Among the proteins identified included vitamin D binding protein (VDBP), aldolase A (aldo A), and apolipoproteinE (apoE). Immunoblotting results validated a significant reduction in these proteins in the plasma at 2 or 5 weeks after vascular injury, in comparison to control animals without vascular injury. These findings suggest that VDBP, aldo A, and apoE may be biomarkers for vascular injury, which will have important prognostic and diagnostic implications.
View details for PubMedID 21416056
A matrix micropatterning platform for cell localization and stem cell fate determination
2010; 6 (12): 4614-4621
To study the role of cell-extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination.
View details for DOI 10.1016/j.actbio.2010.06.033
View details for Web of Science ID 000284385300018
View details for PubMedID 20601236
Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells
STEM CELLS AND DEVELOPMENT
2010; 19 (10): 1617-1625
Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (L-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, L-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of L-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of L-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by L-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function.
View details for DOI 10.1089/scd.2009.0417
View details for Web of Science ID 000282394400014
View details for PubMedID 20064011
Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
2010; 30 (5): 984-U224
We examined the effect of delivery modality on the survival, localization, and functional effects of exogenously administered embryonic stem cells (ESCs) or endothelial cells derived from them (ESC-ECs) in the ischemic hindlimb.Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngeneic murine model of limb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intrafemoral artery (IA), or intrafemoral vein injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by immunohistochemistry, and functional improvement was assessed by laser Doppler perfusion. BLI showed that ESCs localized to the ischemic limb after IM or IA, but not after intrafemoral vein administration. Regardless of the route of administration, ESCs were detected outside the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion and generated teratomas. In contrast, ESC-ECs delivered by all 3 modalities localized to the ischemic limb, as assessed by BLI. Most surprisingly, ESC-EC injected intrafemoral vein eventually localized to the ischemic limb after initially lodging in the pulmonary circulation. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Notably, ESC-ECs were not detected in the spleen or lungs after 2 weeks, regardless of route of administration. Furthermore, ESC-ECs significantly improved limb perfusion and neovascularization compared with the parental ESCs or the vehicle control group.In contrast to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb by IA, IM, and intrafemoral vein delivery. ESC-ECs engrafted into the ischemic microvasculature, enhanced neovascularization, and improved limb perfusion.
View details for DOI 10.1161/ATVBAHA.110.202796
View details for Web of Science ID 000276677700015
View details for PubMedID 20167654
Bone marrow-derived mesenchymal stem cells in fibrin augment angiogenesis in the chronically infarcted myocardium
2009; 4 (4): 527-538
Current efforts to treat myocardial infarction include the delivery of cells and matrix scaffolds. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells that secrete angiogenic growth factors, and fibrin has been shown to be a biomaterial that provides structural support to cells and tissues. The objective of this study was to characterize the attachment and viability of BM-MSCs in fibrin in vitro, and then to assess the efficacy of treatment with BM-MSCs in fibrin for promoting neovascularization in the chronically infarcted myocardium.BM-MSCs were cultured in fibrin and assessed for cell attachment and viability by using immunofluorescence staining for actin filaments and Live/Dead((R)) viability assays, respectively. To determine the efficacy of BM-MSCs in fibrin in vivo, chronically infarcted rat hearts were treated with either cells, cells in fibrin, fibrin or saline (n = 9). After 5 weeks, the infarct scar tissues were assessed for neovascularization.BM-MSCs exhibited robust cell attachment and viability when cultured in fibrin in vitro. Furthermore, when injected together into the infarcted tissue, BM-MSCs in fibrin could enhance neovasculature formation by increasing capillary density, in comparison to treatment by cells or fibrin separately. Concomitant to significant improvement in capillary density was an increase in the levels of VEGF in the infarct scar.This study demonstrates the angiogenic potential of the combined delivery of BM-MSCs and fibrin, and highlights the advantage of stem cell-matrix approaches for myocardial repair.
View details for DOI 10.2217/RME.09.32
View details for Web of Science ID 000268122400010
View details for PubMedID 19580402
Embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia.
Journal of visualized experiments : JoVE
Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD. In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.
View details for DOI 10.3791/1034
View details for PubMedID 19229180
Murine model of hindlimb ischemia.
Journal of visualized experiments : JoVE
In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.
View details for DOI 10.3791/1035
View details for PubMedID 19229179
Mesenchymal stem cells for vascular regeneration
2008; 3 (6): 877-892
Mesenchymal stem cells (MSCs) have tremendous potential for regenerative medicine, and have been researched for the treatment of cardiovascular diseases. MSCs are a promising cell type because of their ease of isolation and expansion, their multipotency and their low immunogenicity. However, in order to fully utilize the therapeutic potential of MSCs, it is important to understand the intrinsic property of MSCs and the role of the microenvironment in modulating MSC behavior and function. Microenvironmental factors such as mechanical cues, soluble factors and matrix properties not only regulate MSC differentiation, but also modulate MSC signaling to the surrounding environment. Understanding the properties of MSCs and the role of the microenvironment will be beneficial for developing in vivo therapies for the construction of tissue-engineered vascular grafts and the treatment of ischemic cardiac tissues.
View details for DOI 10.2217/17460718.104.22.1687
View details for Web of Science ID 000261008000015
View details for PubMedID 18947310
Mechanobiology of mesenchymal stem cells and their use in cardiovascular repair
FRONTIERS IN BIOSCIENCE-LANDMARK
2007; 12: 5098-5116
Mesenchymal stem cells (MSCs) derived from bone marrow have shown great promise in tissue repair. While these cells induce little immune response, they show marked self-renewal properties and can differentiate into many cell types. Recent evidence shows that mechanical factors such as fluid shear stress, mechanical strain and the rigidity of extracellular matrix can regulate the proliferation and differentiation of MSCs through various signaling pathways. Transplanted MSCs enhance angiogenesis and contribute to remodeling of the vasculature. In this review, we will focus on the responses of vascular cells and MSCs to shear stress, strain and matrix rigidity and will discuss the use of MSCs in myocardial repair and vascular tissue engineering.
View details for DOI 10.2741/2551
View details for Web of Science ID 000253943900025
View details for PubMedID 17569633
Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering
2007; 13 (8): 1809-1823
The field of cardiovascular tissue engineering has experienced tremendous advances in the past several decades, but the clinical reality of engineered heart tissue and vascular conduits remains immature. Stem cells and progenitor cells are promising cell sources for engineering functional cardiovascular tissues. To realize the therapeutic potential of stem cells and progenitor cells, we need to understand how microenvironmental cues modulate and guide stem cell differentiation and organization. This review describes the current understanding of the chemical and physical regulation of embryonic and adult stem cells for potential applications in cardiovascular repair, focusing on cardiac therapies after myocardial infarction and the engineering of vascular conduits.
View details for DOI 10.1089/ten.2006.0096
View details for Web of Science ID 000248742200003
View details for PubMedID 17518703
Antibody targeting of stem cells to infarcted myocardium
2007; 25 (3): 712-717
Hematopoietic stem cell (HSC) therapy for myocardial repair is limited by the number of stem cells that migrate to, engraft in, and proliferate at sites of injured myocardium. To alleviate this limitation, we studied whether a strategy using a bispecific antibody (BiAb) could target human stem cells specifically to injured myocardium and preserve myocardial function. Using a xenogeneic rat model whereby ischemic injury was induced by transient ligation of the left anterior descending artery (LAD), we determined the ability of a bispecific antibody to target human CD34+ cells to specific antigens expressed in ischemic injured myocardium. A bispecific antibody comprising an anti-CD45 antibody recognizing the common leukocyte antigen found on HSCs and an antibody recognizing myosin light chain, an organ-specific injury antigen expressed by infarcted myocardium, was prepared by chemical conjugation. CD34+ cells armed and unarmed with this BiAb were injected intravenously in rats 2 days postmyocardial injury. Immunohistochemistry studies showed that the armed CD34+ cells specifically localized to the infarcted region of the heart, colocalized with troponin T-stained cells, and colocalization with vascular structures. Compared to unarmed CD34+ cells, the bispecific antibody improved delivery of the stem cells to injured myocardium, and such targeted delivery was correlated with improved myocardial function 5 weeks after infarction (p < .01). Bispecific antibody targeting offers a unique means to improve the delivery of stem cells to facilitate organ repair and a tool to study stem cell biology.
View details for DOI 10.1634/stemcells.2005-0602
View details for Web of Science ID 000244847100020
View details for PubMedID 17138964
Myotube assembly on nanofibrous and micropatterned polymers
2006; 6 (3): 537-542
Skeletal muscle consists of parallel bundles of myotubes formed by the fusion of myoblasts. We fabricated nanofibrous and micropatterned polymers as cell culture substrates to guide the morphogenesis of muscular tissue. The nanoscale and microscale topographic features regulate cell and cytoskeleton alignment, myotube assembly, myotube striation, and myoblast proliferation. This bottom-up approach from nanoscale to tissue level demonstrates the potential of nanofibrous polymers for engineering the assembly of cell and tissue structure.
View details for DOI 10.1021/nl060060o
View details for Web of Science ID 000236049800040
View details for PubMedID 16522058
A rodent model of myocardial infarction for testing the efficacy of cells and polymers for myocardial reconstruction
2006; 1 (3): 1596-1609
We have developed a robust rat model of myocardial infarction (MI). Here we describe the step-by-step protocol for creating an ischemia-reperfusion rat model of MI. We also describe how to deliver therapeutic injections of mesenchymal stem cells (MSCs) together with fibrin, to show an application of this model. In addition, to confirm the presence of fibrin and cells in the infarct, visualization of MSCs and fibrin by histological techniques are also described. The ischemia-reperfusion MI model can be modified and generalized for use with various injectable polymers, cell types, drugs, DNA and combinations thereof. The model can be created in 7 days or less, depending on the timing of therapeutic intervention.
View details for DOI 10.1038/nprot.2006.188
View details for Web of Science ID 000251155400065
View details for PubMedID 17406452
Mechanotransduction in endothelial cell migration
JOURNAL OF CELLULAR BIOCHEMISTRY
2005; 96 (6): 1110-1126
The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.
View details for DOI 10.1002/jcb.20614
View details for Web of Science ID 000233353000002
View details for PubMedID 16167340
Injectable biopolymers enhance angiogenesis after myocardial infarction
2005; 11 (11-12): 1860-1866
Novel strategies by which to repair ischemic myocardium after myocardial infarction include the use of three-dimensional polymer scaffolds. A comparative study was carried out to assess the therapeutic potential of fibrin, collagen I, and Matrigel as injectable biopolymers for repair after myocardial infarction. Using a rat model of left coronary artery occlusion followed by reperfusion, local injection of the biopolymers into the infarct zone yielded significantly higher levels of capillary formation, when compared with the saline control group, at 5 weeks posttreatment. However, the degree of angiogenesis was not significantly different among the biopolymers. In addition, the collagen biopolymer significantly enhanced infiltration of myofibroblasts into the infarct area when compared with the control group. The results of this study highlight the potential clinical benefit of these biopolymers as injectable scaffolds or cell delivery vehicles to the infarct zone after infarction.
View details for Web of Science ID 000234829500025
View details for PubMedID 16411832
Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides
2004; 75 (1): 71-84
A comprehensive study of the base hydrolysis of all phosphodiester bond-containing capsular polysaccharides of the 23-valent pneumococcal vaccine is described here. Capsular polysaccharides from serotypes 6B, 10A, 17F, 19A, 19F, and 20 contain a phosphodiester bond that connects the repeating units in these polysaccharides (also referred to as backbone phosphodiester bonds), and polysaccharides from serotypes 11A, 15B, 18C, and 23F contain a phosphodiester bond that links a side chain to their repeating units. Molecular weight measurements of the polysaccharides, using high performance size exclusion chromatography with tandem multiangle laser light scattering and refractive index detection, was used to evaluate the kinetics of hydrolysis. The measurement of molecular weight provides a high degree of sensitivity in the case of small extents of reaction, thus allowing reliable measurements of the kinetics over short times. Pseudo-first-order rate constants for these polysaccharides were estimated using a simple model that accounts for the polydispersity of the starting sample. It was found that the relative order of backbone phosphodiester bond instability due to base hydrolysis was 19A > 10A > 19F > 6B > 17F, 20. Degradation of side-chain phosphodiester bonds was not observed, although the high degree of sensitivity in measurements is lost in this case, due to the low contribution of the side chains to the total polysaccharide molecular weight. In comparison with literature data on pneumococcal polysaccharide 6A, 19A was found to be the more labile, and hence appears to be the most labile pneumococcal polysaccharide studied to date. The rate of hydrolysis increased at higher pH and in the presence of divalent cation, but the extent was lower than expected based on similar data on RNA. Finally, the differences in the phosphodiester bond stabilities were analyzed by considering stereochemical factors in these polysaccharides. These results also provide a framework for evaluation of molecular integrity of phosphodiester-bond-containing polysaccharides in different solution conditions.
View details for DOI 10.1002/bip.20087
View details for Web of Science ID 000223580500005
View details for PubMedID 15307199
Tissue engineering of muscle on micropatterned polymer films.
Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
2004; 7: 4966-4969
Tissue engineered skeletal muscle has potential physiologically relevant environments to study myogenesis and investigate the organization, differentiation and proliferation to be used for the therapy of muscular dysfunction. In order to engineer skeletal muscle that better resemble the structured architecture in vivo, we cultured myoblasts on topographically micropatterned elastic polymer films with 10-mum wide microgrooves. The organization and differentiation of myoblasts on nonpatterned and micropatterned PDMS films were characterized. In comparison to the myoblasts on nonpatterned PDMS films, myoblasts on micropatterned PDMS films aligned themselves along the direction of the microgrooves. The myoblasts on micropatterned films formed long and unbranched myotubes that had uniform diameter and aligned in the microgroove direction, suggesting that microgrooves promote end-to end fusion of myoblasts; in contrast, myotubes formed on nonpatterned surface were short and less uniform in diameter, and oriented in various directions. This study demonstrates a new approach to engineer muscular tissues on flexible substrate, and highlights the importance of topographical cues for creating more engineer skeletal muscle.
View details for PubMedID 17271429
Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2003; 100 (22): 12741-12746
Human embryonic stem (hES) cells hold promise as an unlimited source of cells for transplantation therapies. However, control of their proliferation and differentiation into complex, viable 3D tissues is challenging. Here we examine the use of biodegradable polymer scaffolds for promoting hES cell growth and differentiation and formation of 3D structures. We show that complex structures with features of various committed embryonic tissues can be generated, in vitro, by using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as poly(lactic-co-glycolic acid)/poly(L-lactic acid) polymer scaffolds. We found that hES cell differentiation and organization can be influenced by the scaffold and directed by growth factors such as retinoic acid, transforming growth factor beta, activin-A, or insulin-like growth factor. These growth factors induced differentiation into 3D structures with characteristics of developing neural tissues, cartilage, or liver, respectively. In addition, formation of a 3D vessel-like network was observed. When transplanted into severe combined immunodeficient mice, the constructs continue to express specific human proteins in defined differentiated structures and appear to recruit and anastamose with the host vasculature. This approach provides a unique culture system for addressing questions in cell and developmental biology, and provides a potential mechanism for creating viable human tissue structures for therapeutic applications.
View details for DOI 10.1073/pnas.1735463100
View details for Web of Science ID 000186301100043
View details for PubMedID 14561891
Regulation of vascular smooth muscle cells by micropatterning
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2003; 307 (4): 883-890
Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.
View details for DOI 10.1016/S0006-291X(03)01285-3
View details for Web of Science ID 000184510100021
View details for PubMedID 12878194
Detection of numerical chromosomal abnormalities in epithelial ovarian neoplasms by fluorescence in situ hybridization (Fish) and a review of the current literature
APPLIED IMMUNOHISTOCHEMISTRY & MOLECULAR MORPHOLOGY
2002; 10 (2): 187-193
Preliminary retrospective chromosomal analysis was performed using fluorescence in situ hybridization (FISH) with alphoid DNA probes for chromosomes 1, 3, 6, 8, 12, 17, and X. Twenty-four epithelial ovarian tumors were examined in this pilot study, including 8 borderline (LMP) serous tumors, 9 serous carcinoma, and 7 mucinous carcinoma. Hybridization signals were counted to demonstrate the frequency of aneusomy, trace chromosomal progression, and identify the predominance of chromosome copy number abnormalities that are specific to a particular histotype. The preliminary results revealed almost an equal number of mean aneusomies in serous (58.13 +/- 13%) and mucinous (64.33 +/- 10%) carcinoma, both of which were slightly higher than borderline serous tumors (50.57 +/- 17%). Hyposomies 3 and X were significantly higher in mucinous than in serous ovarian carcinomas, and lowest in borderline serous tumors (P<0.05 and P<0.01). Signal losses were a more frequent abnormality in all three histologic subtypes. Mucinous carcinomas showed a loss of chromosomes 8 (45.00 +/- 28%) and 3 (43.14 +/- 16%), in addition to a loss of chromosome X (56.29 +/- 12%). Serous carcinomas showed a gain of chromosome 1 (39.44 +/- 32%), followed by losses of chromosomes 6 (37.00 +/- 20%), 17 (36.44 +/- 19%), and 8 (36.89 +/- 19%). In borderline serous tumors, the most frequent findings were losses of chromosomes 6 (38.00 +/- 17%), 12 (36.88 +/- 17%), and 3 (36.13 +/- 21%). However, further research is necessary to substantiate these preliminary results and elucidate their clinical significance. A brief review of the literature pertaining to interphase cytogenetics in ovarian epithelial tumors is discussed also.
View details for Web of Science ID 000175774500016
View details for PubMedID 12051640
Optimization of fluorescence in situ hybridization (FISH) in cytological specimens.
Cell vision : the journal of analytical morphology
1998; 5 (1): 85-86
View details for PubMedID 9660738