Publications by authors named "Anton V Borovjagin"

31 Publications

Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction.

Circulation 2021 Jul 6;144(3):210-228. Epub 2021 May 6.

Department of Biomedical Engineering (M.Z., Y.N., Y.W., W.B., A.V.B., Y.Z., G.P.W., J.Z.), the University of Alabama at Birmingham.

Background: Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2CMs or CCND2CMs, respectively) and injected into infarcted pig hearts.

Methods: Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2CMs or CCND2CMs (3×10 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area.

Results: The number of the engrafted CCND2CMs exceeded that of the engrafted CCND2CMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2CMs or the delivery vehicle, the administration of CCND2CM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2CM-treated than from the CCND2CM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2CMs (CCND2Exos) than from the CCND2CMs (CCND2Exos). As demonstrated by our study, CCND2Exos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2Exos versus CCND2Exos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis.

Conclusions: We demonstrated that enhanced potency of CCND2CMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2CMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.120.049497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292228PMC
July 2021

Individualized Surgical Reconstruction of the Right Ventricle Outflow Tract in Double Outlet Right Ventricle With Mirror Image-Dextrocardia.

Front Pediatr 2021 19;9:611007. Epub 2021 Feb 19.

Department of Cardiovascular Surgery, The Second Xiangya Hospital of Central South University, Changsha, China.

The purpose of this study was to report our experience in the surgical reconstruction of the right ventricular outflow tract in double outlet right ventricle with a major coronary artery crossing the right ventricular outflow tract in the presence of mirror image-dextrocardia. From January 2005 to December 2019, 19 double outlet right ventricle patients (median age 4 years) with mirror image-dextrocardia and a major coronary artery crossing the right ventricular outflow tract received surgical repair. An autologous pericardial patch was used to enlarge the right ventricular outflow tract in four patients without pulmonary stenosis and three patients with mild pulmonary stenosis. A valved bovine jugular venous conduit was added to a hypoplastic native pathway in nine patients, among which six patients with moderate pulmonary stenosis received small-sized bovine jugular venous conduit implantation (diameter ≤ 16 mm). In comparison, a large-sized bovine jugular venous conduit (diameter >16 mm) was adopted in a total of three patients with severe pulmonary stenosis. Finally, three patients with preoperative pulmonary hypertension (mean pulmonary artery pressure ≥40 mmHg) did not undergo further intervention of right ventricular outflow tract due to the adequate outflow tract blood flow. There was no hospital mortality. One patient with sub-pulmonary ventricular septal defect and concomitant severe pulmonary hypertension died from respiratory failure 11 months after the operation. Kaplan-Meier survival was 94% at 5, 10 years. Within a mean echocardiographic follow-up of 6.9 ± 3.6 years, a total of two patients received reintervention due to valvular stenosis of the bovine jugular venous conduit (pressure gradient > 50 mmHg at 4 and 9 years) after surgical operation. Actuarial freedom from reoperation was 90 and 72% at 5 and 10 years, respectively. During the last echocardiographic follow-up phase, all the survivors were in NYHA class I. Double outlet right ventricle with mirror image-dextrocardia is a rare and complicated congenital cardiac malformation. Surgical reconstruction of the right ventricular outflow tract should be individualized based on the degree of pulmonary stenosis and the specific anatomical features of each patient. Reconstructing the pulmonary artery using the various sizes of valved bovine jugular venous conduit is a safe and effective surgical method.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fped.2021.611007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933223PMC
February 2021

Calreticulin is important for the development of renal fibrosis and dysfunction in diabetic nephropathy.

Matrix Biol Plus 2020 Nov 3;8:100034. Epub 2020 Apr 3.

Department of Pathology, University of Alabama at Birmingham, Birmingham, AL35294-0019, USA.

Previously, our lab showed that the endoplasmic reticulum (ER) and calcium regulatory protein, calreticulin (CRT), is important for collagen transcription, secretion, and assembly into the extracellular matrix (ECM) and that ER CRT is critical for TGF-β stimulation of type I collagen transcription through stimulation of ER calcium release and NFAT activation. Diabetes is the leading cause of end stage renal disease. TGF-β is a key factor in the pathogenesis of diabetic nephropathy. However, the role of calreticulin () in fibrosis of diabetic nephropathy has not been investigated. In current work, we used both in vitro and in vivo approaches to assess the role of ER CRT in TGF-β and glucose stimulated ECM production by renal tubule cells and in diabetic mice. Knockdown of by siRNA in a human proximal tubular cell line (HK-2) showed reduced induction of soluble collagen when stimulated by TGF-β or high glucose as compared to control cells, as well as a reduction in fibronectin and collagen IV transcript levels. CRT protein is increased in kidneys of mice made diabetic with streptozotocin and subjected to uninephrectomy to accelerate renal tubular injury as compared to controls. We used renal-targeted ultrasound delivery of Cre-recombinase plasmid to knockdown specifically CRT expression in the remaining kidney of uninephrectomized mice with streptozotocin-induced diabetes. This approach reduced CRT expression in the kidney, primarily in the tubular epithelium, by 30-55%, which persisted over the course of the studies. Renal function as measured by the urinary albumin/creatinine ratio was improved in the mice with knockdown of CRT as compared to diabetic mice injected with saline or subjected to ultrasound and injected with control GFP plasmid. PAS staining of kidneys and immunohistochemical analyses of collagen types I and IV show reduced glomerular and tubulointerstitial fibrosis. Renal sections from diabetic mice with CRT knockdown showed reduced nuclear NFAT in renal tubules and treatment of diabetic mice with 11R-VIVIT, an NFAT inhibitor, reduced proteinuria and renal fibrosis. These studies identify ER CRT as an important regulator of TGF-β stimulated ECM production in the diabetic kidney, potentially through regulation of NFAT-dependent ECM transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mbplus.2020.100034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852315PMC
November 2020

KISS1 in breast cancer progression and autophagy.

Cancer Metastasis Rev 2019 09;38(3):493-506

Department of Cancer Biology, University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, KS, 66160, USA.

Tumor suppressors are cellular proteins typically expressed in normal (non-cancer) cells that not only regulate such cellular functions as proliferation, migration and adhesion, but can also be secreted into extracellular space and serve as biomarkers for pathological conditions or tumor progression. KISS1, a precursor for several shorter peptides, known as metastin (Kisspeptin-54), Kisspeptin-14, Kisspeptin-13 and Kisspeptin-10, is one of those metastasis suppressor proteins, whose expression is commonly downregulated in the metastatic tumors of various origins. The commonly accepted role of KISS1 in metastatic tumor progression mechanism is the ability of this protein to suppress colonization of disseminated cancer cells in distant organs critical for the formation of the secondary tumor foci. Besides, recent evidence suggests involvement of KISS1 in the mechanisms of tumor angiogenesis, autophagy and apoptosis regulation, suggesting a possible role in both restricting and promoting cancer cell invasion. Here, we discuss the role of KISS1 in regulating metastases, the link between KISS1 expression and the autophagy-related biology of cancer cells and the perspectives of using KISS1 as a potential diagnostic marker for cancer progression as well as a new anti-cancer therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10555-019-09814-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986448PMC
September 2019

Reabsorbable Pins can Reinforce an Early Sternal Stability After Median Sternotomy in Young Children with Congenital Heart Disease.

Pediatr Cardiol 2019 Dec 23;40(8):1728-1734. Epub 2019 Sep 23.

Department of Cardiovascular Surgery, The Second Xiangya Hospital, Central South University, Middle Renmin Road 139, Changsha, 410011, China.

We evaluated the efficacy of bioresorbable sternal reinforcement device (poly-L-lactide sternal pins) on sternal healing after median sternotomy in young children (with body weight less than 10 kg) with congenital heart disease (CHD). Data from 85 patients, who underwent CHD surgery through median sternotomy from October 2016 to May 2018, were collected and analyzed. Sternal pins were utilized in 85 patients (10 mm × 1 mm × 1 mm for patients with body weights less than 5 kg and 15 mm × 2 mm × 2 mm for those weighing between 5 and 10 kg) in addition to sternum closure with Ethicon PDSII running sutures (Group A), while 84 patients received the Ethicon sternal closure (Group B) with no pins. The occurrence of sternal dehiscence, anterior-posterior displacement, and high-low displacement was evaluated by physical examination and three-dimensional computed tomography at one month postoperatively. No anterior-posterior sternal displacement (0%) was observed in Group A, while 10 anterior-posterior displacements (11.9%) were observed in Group B (P < 0.01). The number of sternal caudal-cranial displacements in Groups A and B was 4 (4.71%) and 5 (5.35%), respectively (P = 0.870). While no sternal dehiscence (0%) was observed in Group A, 7 out of 84 patients (8.33%) in Group B exhibited obvious sternal dehiscence (P < 0.01). The bioresorbable poly-L-lactide sternal pins reduced an anterior-posterior sternal displacement and sternal dehiscence, which was accompanied by a significant improvement of an early sternal fixation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00246-019-02212-1DOI Listing
December 2019

Genetic strategy to decrease complement activation with adenoviral therapies.

PLoS One 2019 26;14(4):e0215226. Epub 2019 Apr 26.

Department of Otolaryngology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

Background: A major obstacle to using recombinant adenoviral vectors in gene therapy is the natural ability of human adenovirus to activate the classical and alternate complement pathways. These innate immune responses contribute to hepatic adenoviral uptake following systemic delivery and enhance the humoral immune responses associated with adenoviral infection.

Methods: A recombinant Ad5 vector was genetically modified to display a peptide sequence ("rH17d'"), a known inhibitor of the classical complement pathway. The replication-defective vectors Ad5.HVR2-rH17d' and Ad5.HVR5-rH17d' were constructed by engineering the rH17d' peptide into the hypervariable region (HVR)-2 or HVR5 of their major capsid protein hexon. Control Ad5 vectors were created by incorporation of a 6-histidine (His6)-insert in either HVR2 or HVR5 (Ad5.HVR2-His6 and Ad5.HVR5-His6, respectively). All vectors encoded CMV promoter-controlled firefly luciferase (Luc). The four vectors were evaluated in TIB76 mouse liver cells and immunocompetent mice to compare infectivity and liver sequestration, respectively.

Results: In vitro studies demonstrated that preincubation of all the Ad5 vectors with fresh serum significantly increased their gene transfer relative to preincubation with PBS except Ad5.HVR5-rH17d', whose infectivity of liver cells showed no serum-mediated enhancement. In line with that, mice injected with Ad5.HVR2-rH17d' or Ad5.HVR5-rH17d' showed significantly lower luciferase expression levels in the liver as compared to the respective control vectors, whereas efficiency of tumor transduction by rH17d' and His6 vectors following their intratumoral injection was similar.

Conclusions: Displaying a complement-inhibiting peptide on the Ad5 capsid surface by genetic modification of the hexon protein could be a suitable strategy for reducing Ad5 liver tropism (Ad5 sequestration by liver), which may be applicable to other gene therapy vectors with natural liver tropism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215226PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485611PMC
February 2020

Y-27632 preconditioning enhances transplantation of human-induced pluripotent stem cell-derived cardiomyocytes in myocardial infarction mice.

Cardiovasc Res 2019 02;115(2):343-356

Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham, 1670 University Blvd, VH G094E, Birmingham, AL, USA.

Aims: The effectiveness of cell-based treatments for regenerative myocardial therapy is limited by low rates of cell engraftment. Y-27632 inhibits Rho-associated protein kinase (ROCK), which regulates the cytoskeletal changes associated with cell adhesion, and has been used to protect cultured cells during their passaging. Here, we investigated whether preconditioning of cardiomyocytes, derived from human-induced pluripotent stem cells (hiPSC-CM), with Y-27632 improves their survival and engraftment in a murine model of acute myocardial infarction (MI).

Methods And Results: After MI induction, mice were subjected to intramyocardial injections of phosphate-buffered saline, hiPSC-CM cultured under standard conditions (hiPSC-CM-RI), or Y-27632-preconditioned hiPSC-CM (hiPSC-CM+RI). The resulting engraftment rate calculated 4 weeks after implantation was significantly higher and the abundance of apoptotic transplanted cells was significantly lower in hiPSC-CM+RI recipients than in hiPSC-CM-RI animals. In cultured hiPSC-CM, Y-27632-preconditioning reversibly reduced contractile activity and the expression of troponin genes, while increasing their attachment to an underlying mouse cardiomyocyte (HL1) monolayer. Y-27632 preconditioning also increased the expression of N-cadherin and integrin ß1, the two cell junction proteins. hiPSC-CM+RI were also larger in cell area with greater cytoskeletal alignment and a more rod-like shape than hiPSC-CM-RI, both after transplantation (in vivo) and in culture. The effects of Y-27632 preconditioning on contractile activity and morphology of hiPSC-CMs in culture, as well as on their engraftment rate and apoptotic death in MI mouse grafts, could be recapitulated by hiPSC-CM treatment with the L-type calcium-channel blocker verapamil.

Conclusion: Preconditioning with the ROCK inhibitor Y-27632 increased the engraftment of transplanted hiPSC-CM in a murine MI model, while reversibly impairing hiPSC-CM contractility and promoting adhesion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvy207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341224PMC
February 2019

Regenerative Potential of Neonatal Porcine Hearts.

Circulation 2018 12;138(24):2809-2816

Department of Biomedical Engineering, School of Medicine, and School of Engineering (W.Z., E.Z., M.Z., C.F., Y.T., J.D.H., A.V.B., G.P.W., G.Q., J.Z.), University of Alabama at Birmingham.

Background: Rodent hearts can regenerate myocardium lost to apical resection or myocardial infarction for up to 7 days after birth, but whether a similar window for myocardial regeneration also exists in large mammals is unknown.

Methods: Acute myocardial infarction (AMI) was surgically induced in neonatal pigs on postnatal days 1, 2, 3, 7, and 14 (ie, the P1, P2, P3, P7, and P14 groups, respectively). Cardiac systolic function was evaluated before AMI and at 30 days post-AMI via transthoracic echocardiography. Cardiomyocyte cell cycle activity was assessed via immunostaining for proliferation and mitosis markers, infarct size was evaluated histologically, and telomerase activity was measured by quantitative polymerase chain reaction.

Results: Systolic function at day 30 post-AMI was largely restored in P1 animals and partially restored in P2 animals, but significantly impaired when AMI was induced on postnatal day 3 or later. Hearts of P1 animals showed little evidence of scar formation or wall thinning on day 30 after AMI, with increased measures of cell-cycle activity seen 6 days after AMI (ie, postnatal day 7) compared with postnatal day 7 in noninfarcted hearts.

Conclusions: The neonatal porcine heart is capable of regeneration after AMI during the first 2 days of life. This phenomenon is associated with induction of cardiomyocyte proliferation and is lost when cardiomyocytes exit the cell cycle shortly after birth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.118.034886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301098PMC
December 2018

Tamoxifen overrides autophagy inhibition in Beclin-1-deficient glioma cells and their resistance to adenovirus-mediated oncolysis via upregulation of PUMA and BAX.

Oncogene 2018 11 10;37(46):6069-6082. Epub 2018 Jul 10.

Northwestern Brain Tumor Institute, Northwestern University, Chicago, IL, 60611, USA.

Autophagy is an evolutionarily conserved process regulating cellular homeostasis via digestion of dysfunctional proteins and whole cellular organelles by mechanisms, involving their enclosure into double-membrane vacuoles that are subsequently fused to lysosomes. Glioma stem cells utilize autophagy as a main mechanism of cell survival and stress response. Most recently, we and others demonstrated induction of autophagy in gliomas in response to treatment with chemical drugs, such as temozolomide (TMZ) or oncolytic adenoviruses (Ads). As autophagy has been implicated in the mechanism of Ad-mediated cell killing, autophagy deficiency in some glioma tumors could be the reason for their resistance to oncolysis. Despite the observed connection, the exact relationship between autophagy-activating cell signaling and adenoviral infection remains unclear. Here, we report that inhibition of autophagy in target glioma cells induces their resistance to killing by oncolytic agent CRAd-S-5/3. Furthermore, we found that downregulation of autophagy inducer Beclin-1 inhibits replication-competent Ad-induced oncolysis of human glioma by suppressing cell proliferation and inducing premature senescence. To overcome the autophagy-deficient state of such glioma cells and restore their susceptibility to oncolytic Ad infection, we propose treating glioma tumors with an anticancer drug tamoxifen (TAM) as a means to induce apoptosis in Ad-targeted cancer cells via upregulation of BAX/PUMA genes. In agreement with the above hypothesis, our data suggest that TAM improves susceptibility of Beclin-1-deficient glioma cells to CRAd-S-5/3 oncolysis by means of activating autophagy and pro-apoptotic signaling pathways in the target cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-018-0395-9DOI Listing
November 2018

Spheroids of cardiomyocytes derived from human-induced pluripotent stem cells improve recovery from myocardial injury in mice.

Am J Physiol Heart Circ Physiol 2018 08 6;315(2):H327-H339. Epub 2018 Apr 6.

Department of Biomedical Engineering, School of Medicine, School of Engineering, The University of Alabama at Birmingham , Birmingham, Alabama.

The microenvironment of native heart tissue may be better replicated when cardiomyocytes are cultured in three-dimensional clusters (i.e., spheroids) than in monolayers or as individual cells. Thus, we differentiated human cardiac lineage-induced pluripotent stem cells in cardiomyocytes (hiPSC-CMs) and allowed them to form spheroids and spheroid fusions that were characterized in vitro and evaluated in mice after experimentally induced myocardial infarction (MI). Synchronized contractions were observed within 24 h of spheroid formation, and optical mapping experiments confirmed the presence of both Ca transients and propagating action potentials. In spheroid fusions, the intraspheroid conduction velocity was 7.0 ± 3.8 cm/s on days 1- 2 after formation, whereas the conduction velocity between spheroids increased significantly ( P = 0.003) from 0.8 ± 1.1 cm/s on days 1- 2 to 3.3 ± 1.4 cm/s on day 7. For the murine MI model, five-spheroid fusions (200,000 hiPSC-CMs/spheroid) were embedded in a fibrin patch and the patch was transplanted over the site of infarction. Later (4 wk), echocardiographic measurements of left ventricular ejection fraction and fractional shortening were significantly greater in patch-treated animals than in animals that recovered without the patch, and the engraftment rate was 25.6% or 30% when evaluated histologically or via bioluminescence imaging, respectively. The exosomes released from the spheroid patch seemed to increase cardiac function. In conclusion, our results established the feasibility of using hiPSC-CM spheroids and spheroid fusions for cardiac tissue engineering, and, when fibrin patches containing hiPSC-CM spheroid fusions were evaluated in a murine MI model, the engraftment rate was much higher than the rates we have achieved via the direct intramyocardial injection. NEW & NOTEWORTHY Spheroids fuse in culture to produce structures with uniformly distributed cells. Furthermore, human cardiac lineage-induced pluripotent stem cells in cardiomyocytes in adjacent fused spheroids became electromechanically coupled as the fusions matured in vitro, and when the spheroids were combined with a biological matrix and administered as a patch over the infarcted region of mouse hearts, the engraftment rate exceeded 25%, and the treatment was associated with significant improvements in cardiac function via a paracrine mechanism, where exosomes released from the spheroid patch.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpheart.00688.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139622PMC
August 2018

KISS1 tumor suppressor restricts angiogenesis of breast cancer brain metastases and sensitizes them to oncolytic virotherapy in vitro.

Cancer Lett 2018 03 18;417:75-88. Epub 2017 Dec 18.

Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Moscow, 119991, Russia. Electronic address:

KISS1 tumor suppressor protein regulates cancer cell invasion via MMP9 metalloproteinase. Downregulation of KISS1 gene expression promotes progression of breast cancer and melanoma, resulting in the development of distant metastases. In the current study, we investigated whether restoration of KISS1 expression in KISS1-deficient human metastatic breast cancer cells holds potential as an advanced anticancer strategy. To this end we engineered an infectivity-enhanced conditionally-replicative human adenovirus type 5 encoding KISS1 as an "arming" transgene in the Ad5 E3 region for an ectopic KISS1 expression in transduced cancer cells. The oncolytic potential of the vector was examined using brain-invading metastatic clones of CN34 and MDA-MB-231 breast cancer cells, which supported high levels of AdKISS1 replication, correlating with a robust CRAd-mediated cytotoxicity. Secretion of cellular factors responsible for tumor angiogenesis, cell-to-cell communication and anti-tumoral immune responses upon KISS1 expression in breast cancer cells was analyzed by a RayBiotech Kiloplex Quantibody array. Overall, our results indicate that KISS1 transgene expression provides an important benefit for CRAd-mediated cytotoxicity in breast cancer cells and holds potential as an anticancer treatment in conjunction with oncolytic virotherapy of breast and other metastatic cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.canlet.2017.12.024DOI Listing
March 2018

Large Cardiac Muscle Patches Engineered From Human Induced-Pluripotent Stem Cell-Derived Cardiac Cells Improve Recovery From Myocardial Infarction in Swine.

Circulation 2018 04 12;137(16):1712-1730. Epub 2017 Dec 12.

Department of Biomedical Engineering, School of Medicine and School of Engineering, University of Alabama at Birmingham (L.G., W.Z., S.M., Y.O., X.LO., R.K., A.V.B., G.P.W., A.E.P., V.G.F., S.G.L., J.Z.).

Background: Here, we generated human cardiac muscle patches (hCMPs) of clinically relevant dimensions (4 cm × 2 cm × 1.25 mm) by suspending cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human induced-pluripotent stem cells in a fibrin scaffold and then culturing the construct on a dynamic (rocking) platform.

Methods: In vitro assessments of hCMPs suggest maturation in response to dynamic culture stimulation. In vivo assessments were conducted in a porcine model of myocardial infarction (MI). Animal groups included: MI hearts treated with 2 hCMPs (MI+hCMP, n=13), MI hearts treated with 2 cell-free open fibrin patches (n=14), or MI hearts with neither experimental patch (n=15); a fourth group of animals underwent sham surgery (Sham, n=8). Cardiac function and infarct size were evaluated by MRI, arrhythmia incidence by implanted loop recorders, and the engraftment rate by calculation of quantitative polymerase chain reaction measurements of expression of the human Y chromosome. Additional studies examined the myocardial protein expression profile changes and potential mechanisms of action that related to exosomes from the cell patch.

Results: The hCMPs began to beat synchronously within 1 day of fabrication, and after 7 days of dynamic culture stimulation, in vitro assessments indicated the mechanisms related to the improvements in electronic mechanical coupling, calcium-handling, and force generation, suggesting a maturation process during the dynamic culture. The engraftment rate was 10.9±1.8% at 4 weeks after the transplantation. The hCMP transplantation was associated with significant improvements in left ventricular function, infarct size, myocardial wall stress, myocardial hypertrophy, and reduced apoptosis in the periscar boarder zone myocardium. hCMP transplantation also reversed some MI-associated changes in sarcomeric regulatory protein phosphorylation. The exosomes released from the hCMP appeared to have cytoprotective properties that improved cardiomyocyte survival.

Conclusions: We have fabricated a clinically relevant size of hCMP with trilineage cardiac cells derived from human induced-pluripotent stem cells. The hCMP matures in vitro during 7 days of dynamic culture. Transplantation of this type of hCMP results in significantly reduced infarct size and improvements in cardiac function that are associated with reduction in left ventricular wall stress. The hCMP treatment is not associated with significant changes in arrhythmogenicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.117.030785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5903991PMC
April 2018

VEGF nanoparticles repair the heart after myocardial infarction.

Am J Physiol Heart Circ Physiol 2018 02 3;314(2):H278-H284. Epub 2017 Nov 3.

Department of Biomedical Engineering, School of Medicine and School of Engineering, University of Alabama at Birmingham , Birmingham, Alabama.

Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine that has been shown to promote neovascularization in hearts of patients with ischemic heart disease but can also lead to adverse effects depending on the dose and mode of delivery. We investigated whether prolonged exposure to a low dose of VEGF could be achieved by encapsulating VEGF in polylactic coglycolic acid nanoparticles and whether treatment with VEGF-containing nanoparticles improved cardiac function and protected against left ventricular remodeling in the hearts of mice with experimentally induced myocardial infarction. Polylactic coglycolic acid nanoparticles with a mean diameter of ~113 nm were generated via double emulsion and loaded with VEGF; the encapsulation efficiency was 53.5 ± 1.7% (107.1 ± 3.3 ng VEGF/mg nanoparticles). In culture, VEGF nanoparticles released VEGF continuously for at least 31 days, and in a murine myocardial infarction model, VEGF nanoparticle administration was associated with significantly greater vascular density in the peri-infarct region, reductions in infarct size, and improvements in left ventricular contractile function 4 wk after treatment. Thus, our study provides proof of principle that nanoparticle-mediated delivery increases the angiogenic and therapeutic potency of VEGF for the treatment of ischemic heart disease. NEW & NOTEWORTHY Vascular endothelial growth factor (VEGF) is a well-characterized proangiogenic cytokine but has a short half-life and a rapid clearance rate. When encapsulated in nanoparticles, VEGF was released for 31 days and improved left ventricular function in infarcted mouse hearts. These observations indicate that our new platform increases the therapeutic potency of VEGF.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpheart.00471.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867653PMC
February 2018

Astrocytes promote progression of breast cancer metastases to the brain via a KISS1-mediated autophagy.

Autophagy 2017 5;13(11):1905-1923. Epub 2017 Oct 5.

f Center for Advanced Brain Tumor Treatment , Swedish Neuroscience Institute , Seattle , WA , USA.

Formation of metastases, also known as cancer dissemination, is an important stage of breast cancer (BrCa) development. KISS1 expression is associated with inhibition of metastases development. Recently we have demonstrated that BrCa metastases to the brain exhibit low levels of KISS1 expression at both mRNA and protein levels. By using multicolor immunofluorescence and coculture techniques here we show that normal adult astrocytes in the brain are capable of promoting metastatic transformation of circulating breast cancer cells localized to the brain through secretion of chemokine CXCL12. The latter was found in this study to downregulate KISS1 expression at the post-transcriptional level via induction of microRNA-345 (MIR345). Furthermore, we demonstrated that ectopic expression of KISS1 downregulates ATG5 and ATG7, 2 key modulators of autophagy, and works concurrently with autophagy inhibitors, thereby implicating autophagy in the mechanism of KISS1-mediated BrCa metastatic transformation. We also found that expression of KISS1 in human breast tumor specimens inversely correlates with that of MMP9 and IL8, implicated in the mechanism of metastatic invasion, thereby supporting the role of KISS1 as a potential regulator of BrCa metastatic invasion in the brain. This conclusion is further supported by the ability of KISS1, ectopically overexpressed from an adenoviral vector in MDA-MB-231Br cells with silenced expression of the endogenous gene, to revert invasive phenotype of those cells. Taken together, our results strongly suggest that human adult astrocytes can promote brain invasion of the brain-localized circulating breast cancer cells by upregulating autophagy signaling pathways via the CXCL12-MIR345- KISS1 axis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2017.1360466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788498PMC
June 2019

From Microscale Devices to 3D Printing: Advances in Fabrication of 3D Cardiovascular Tissues.

Circ Res 2017 Jan;120(1):150-165

From the Department of Biomedical Engineering, School of Medicine, School of Engineering, The University of Alabama at Birmingham (A.V.B., J.L.B., J.Z.); and Department of Biomedical Engineering, College of Science and Engineering, The University of Minnesota, Minneapolis (B.M.O.).

Current strategies for engineering cardiovascular cells and tissues have yielded a variety of sophisticated tools for studying disease mechanisms, for development of drug therapies, and for fabrication of tissue equivalents that may have application in future clinical use. These efforts are motivated by the need to extend traditional 2-dimensional (2D) cell culture systems into 3D to more accurately replicate in vivo cell and tissue function of cardiovascular structures. Developments in microscale devices and bioprinted 3D tissues are beginning to supplant traditional 2D cell cultures and preclinical animal studies that have historically been the standard for drug and tissue development. These new approaches lend themselves to patient-specific diagnostics, therapeutics, and tissue regeneration. The emergence of these technologies also carries technical challenges to be met before traditional cell culture and animal testing become obsolete. Successful development and validation of 3D human tissue constructs will provide powerful new paradigms for more cost effective and timely translation of cardiovascular tissue equivalents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.116.308538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5224928PMC
January 2017

MT1-MMP silencing by an shRNA-armed glioma-targeted conditionally replicative adenovirus (CRAd) improves its anti-glioma efficacy in vitro and in vivo.

Cancer Lett 2015 Sep 4;365(2):240-50. Epub 2015 Jun 4.

Center for Advanced Brain Tumor Center, Swedish Neuroscience Institute, 550 17th Avenue, Seattle, WA 98122, USA. Electronic address:

MMP14 (MT1-MMP) is a cell membrane-associated proteinase of the extracellular matrix, whose biological roles vary from angiogenesis to cell proliferation and survival. We recently found a direct correlation between MMP14 expression levels in brain tumors of glioma patients and the disease progression. By using gene silencing as an experimental approach we found that MMP14 knockdown decreases production of pro-angiogenic factors such as VEGF and IL8 and thereby suppresses angiogenesis in glioma tumors. Although the clinical relevance of MMP14 down-regulation and its possible implications for glioma therapy in humans remain unclear, we observed a significant improvement in animal survival upon down-regulation of MMP14 in murine intracranial glioma xenografts infected with MMP14 shRNA-expressing CRAd. We further found that down-regulation of MMP14 in gliomas by combinational treatment with CRAd-S-5/3 and Marimastat, a chemical inhibitor of metalloproteinases, augments suppression of pro-angiogenic factors, caused by the replication-competent adenovirus. We also demonstrated that delivery of MMP14-targeting shRNA by a fiber-modified adenoviral vector to the glioma cells effectively suppresses their proliferation in vitro and in vivo. Thus our data indicate that inhibition of MMP14 expression in tumors in combination with glioma virotherapy could be effectively utilized to suppress angiogenesis and neovascularization of glioma tumors by decreasing production of pro-angiogenic factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.canlet.2015.06.002DOI Listing
September 2015

Therapeutic efficacy of an oncolytic adenovirus containing RGD ligand in minor capsid protein IX and Fiber, Δ24DoubleRGD, in an ovarian cancer model.

J Mol Biochem 2012 Feb;1(1):26-39

Division of Human Gene Therapy, Departments of Medicine, Pathology, Surgery, Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA ; University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

Ovarian cancer is the leading cause of gynecological disease death despite advances in medicine. Therefore, novel strategies are required for ovarian cancer therapy. Conditionally replicative adenoviruses (CRAds), genetically modified as anti-cancer therapeutics, are one of the most attractive candidate agents for cancer therapy. However, a paucity of coxsackie B virus and adenovirus receptor (CAR) expression on the surface of ovarian cancer cells has impeded treatment of ovarian cancer using this approach. This study sought to engineer a CRAd with enhanced oncolytic ability in ovarian cancer cells, "Δ24DoubleRGD." Δ24DoubleRGD carries an arginine-glycine-aspartate (RGD) motif incorporated into both fiber and capsid protein IX (pIX) and its oncolytic efficacy was evaluated in ovarian cancer. analysis of cell viability showed that infection of ovarian cancer cells with Δ24DoubleRGD leads to increased cell killing relative to the control CRAds. Data from this study suggested that not only an increase in number of RGD motifs on the CRAd capsid, but also a change in the repertoir of targeted integrins could lead to enhanced oncolytic potency of Δ24DoubleRGD in ovarian cancer cells . In an intraperitoneal model of ovarian cancer, mice injected with Δ24DoubleRGD showed, however, a similar survival rate as mice treated with control CRAds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755628PMC
February 2012

Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

PLoS One 2011 7;6(10):e24281. Epub 2011 Oct 7.

Department of Periodontics, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America.

To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(v)β3/α(v)β5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024281PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189176PMC
February 2012

Role of RGD-containing ligands in targeting cellular integrins: Applications for ovarian cancer virotherapy (Review).

Exp Ther Med 2010 Mar;1(2):233-240

Division of Human Gene Therapy, Departments of Medicine, Pathology, Surgery, Obstetrics and Gynecology, The Gene Therapy Center, Birmingham, AL, USA.

The purpose of this article was to review the current strategies of targeted therapy to integrins and define the best course of future research in ovarian cancer targeting. Cell surface integrin targeting has been used as a strategy for targeted therapy of several diseases with some success. The combination of virotherapy and integrin-targeting shows promise as a method for targeting ovarian cancer. More specifically, targeting of ovarian cancer with integrin-directed adenoviruses may lead to therapy with fewer toxicities and side effects. This article offers a review of the benefits of integrin-specific targeted therapy for several diseases and proposes a unique anti-ovarian cancer strategy involving the combination of the above with virotherapy as a potential anti-ovarian cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/etm_00000037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008561PMC
March 2010

HIV antigen incorporation within adenovirus hexon hypervariable 2 for a novel HIV vaccine approach.

PLoS One 2010 Jul 27;5(7):e11815. Epub 2010 Jul 27.

Division of Human Gene Therapy, Departments of Medicine, Pathology, Surgery, Obstetrics and Gynecology, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the "antigen capsid-incorporation" strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011815PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2910733PMC
July 2010

A genetic strategy for combined screening and localized imaging of breast cancer.

Mol Imaging Biol 2011 Jun;13(3):452-461

Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA.

Purpose: Improvements are needed for the early detection of breast cancer, as current imaging methods lack sensitivity to detect small tumors and assess their disease phenotype.

Procedures: To address this issue, the dual reporter adenoviral vector (Ad5/3-Id1-SEAP-Id1-mCherry) was produced with a cancer-specific Id1 promoter driving expression of a blood-based screening reporter (secreted embryonic alkaline phosphatase, SEAP) and a fluorescent imaging reporter (mCherry). This diagnostic system was assessed for its screening potential on breast cancer cell lines of various aggressive phenotypes. Reporter expression was measured and correlated with promoter level expression using Western blot. Adenovirus receptor expression was normalized against reporter expression with luciferase infectivity assays. Ad5/3-Id1-SEAP-Id1-mCherry infected MDA-MB-231 cells combined with uninfected cells were implanted into the mammary fat pad of athymic nude mice to recapitulate low-dose tumor delivery. Id1 driven SEAP expression and mCherry imaging were monitored to validate diagnostic sensitivity and efficacy.

Results: Infected breast cancer cell lines displayed SEAP levels in the media that were 10-fold above background by 2 days after infection. Ad5/3-Id1-SEAP-Id1-mCherry infected cells (multiplicity of infection = 10) implanted in athymic nude mice demonstrated a 14-fold increase in serum SEAP levels over baseline when as little as 2.5% of the tumor contained infected cells. This robust response was also found for the mCherry reporter, which was clearly visible in tumor xenografts on day 2 post implantation.

Conclusions: This diagnostic system that combines screening with imaging for early detection and monitoring of breast cancer can be easily extended to other reporters/modalities and cancer-targeting methods. Combining screening with imaging in a genetic, cancer-specific mechanism allows sensitive multi-modal detection and localization of breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11307-010-0377-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359509PMC
June 2011

Noninvasive monitoring of mRFP1- and mCherry-labeled oncolytic adenoviruses in an orthotopic breast cancer model by spectral imaging.

Mol Imaging 2010 Apr;9(2):59-75

Institute of Oral Health Research, University of Alabama at Birmingham School of Dentistry, Birmingham, AL, USA.

Genetic capsid labeling of conditionally replicative adenoviruses (CRAds) with fluorescent tags offers a potentially more accurate monitoring of those virotherapy agents in vivo. The capsid of an infectivity-enhanced CRAd, Ad5/3, delta 24, was genetically labeled with monomeric red fluorescent protein 1 (mRFP1) or its advanced derivative, "mCherry," to evaluate the utility of each red fluorescent reporter and the benefit of CRAd capsid labeling for noninvasive virus tracking in animal tumor models by a new spectral imaging approach. Either reporter was incorporated into the CRAd particles by genetic fusion to the viral capsid protein IX. Following intratumoral injection, localization and replication of each virus in orthotopic breast cancer xenografts were analyzed by spectral imaging and verified by quantitative polymerase chain reaction. Fluorescence in tumors increased up to 2,000-fold by day 4 and persisted for 5 to 7 weeks, showing oscillatory dynamics reflective of CRAd replication cycles. Capsid labeling in conjunction with spectral imaging thus enables direct visualization and quantification of CRAd particles in tumors prior to the reporter transgene expression. This allows for noninvasive control of CRAd delivery and distribution in tumors and facilitates quantitative assessment of viral replication. Although mCherry appeared to be superior to mRFP1 as an imaging tag, both reporters showed utility for CRAd imaging applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952450PMC
April 2010

Novel infectivity-enhanced oncolytic adenovirus with a capsid-incorporated dual-imaging moiety for monitoring virotherapy in ovarian cancer.

Mol Imaging 2009 Sep-Oct;8(5):264-77

Department of Medicine, University of Alabama at Birmingham, USA.

We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for noninvasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk) and monomeric red fluorescent protein 1 (mRFP1) into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2795397PMC
January 2010

Administration of a conditionally replicative oncolytic canine adenovirus in normal dogs.

Cancer Biother Radiopharm 2006 Dec;21(6):601-6

Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA.

Conditionally replicative adenoviruses (CRAds) are engineered to replicate only in the target tissue and destroy tumor through their cytopathic effect. Because of restricted in vivo replication, it is difficult to model behavior of human Ad5-based vectors in animal subjects. To circumvent this, we developed a "syngeneic" canine CRAd based on canine adenovirus type 2 (CAV2) transcriptionally targeted to canine osteosarcoma (OS) cells. Canine OS is an outstanding model of human OS and is the most common primary bone tumor of dogs. Because conventional therapies extend median survival by approximately 6-8 months, canine OS remains a serious therapeutic challenge shared by human OS patients. Prior to using any CRAd for clinical trials in dogs, we sought to examine the effects and safety of administration of OS-targeted CAV2 CRAd in normal dogs. Short-term physiologic indicators of stress and shock, as well as gross and histological changes in a variety of tissues, were examined, and no major signs of virus-associated toxicity were noted. In addition, short-term immunosuppression did not increase CRAd toxicity. This study marks the first administration of a CRAd in an outbred large animal model and is an important milestone in the application of this modality in human patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/cbr.2006.21.601DOI Listing
December 2006

Thermostability/infectivity defect caused by deletion of the core protein V gene in human adenovirus type 5 is rescued by thermo-selectable mutations in the core protein X precursor.

J Mol Biol 2007 Mar 6;366(4):1142-60. Epub 2006 Dec 6.

Division of Human Gene Therapy, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Mastadenoviruses represent one of the four major genera of the Adenoviridae family comprising a variety of mammalian pathogens including human adenovirus (Ad), whose genomes encode a gene for minor core protein V (pV), not found in other genera of Adenoviridae. Deletion of other genus-specific genes (gene IX and E3 genes) from the Ad type 5 (Ad5) genome has been studied experimentally in vitro and the results on biological characterization of the mutants support the phylogenetic evidence of those genes being non-essential for Ad viability. On this basis it seemed logical to suggest that a deletion of gene V from the Ad5 genome could also be tolerated. To test this hypothesis we constructed and rescued the first pV-deletion mutant of human Ad5. As compared to Ad5, this mutant formed small plaques, had dramatically reduced thermostability and lower infectivity. A subsequent thermoselection screen of the pV-deleted Ad5 allowed isolation of a suppressor mutant Ad5-dV/TSB with restored biological characteristics. Since replication and viral assembly of Ad5-dV/TSB could still occur in the absence of pV, we conclude that pV is a non-essential component of the virion. The observed rescue of the biological defects appears to be associated with a cluster of point mutations in the gene encoding the precursor for the other core protein, X/Mu. This finding, thus, suggests possible roles of pV and protein X/Mu precursor in viral assembly. It also provides an interesting insight into genetic events that mediate molecular adaptation of viruses to possible changes in the genetic background in the course of their evolutionary divergence. The possible mechanism of the observed genetic suppression is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2006.11.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2203208PMC
March 2007

An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA.

Nucleic Acids Res 2005 6;33(15):4995-5005. Epub 2005 Sep 6.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Division of Biology and Medicine, Providence, RI 02912, USA.

Correct docking of U3 small nucleolar RNA (snoRNA) on pre-ribosomal RNA (pre-rRNA) is essential for rRNA processing to produce 18S rRNA. In this report, we have used Xenopus oocytes to characterize the structural requirements of the U3 snoRNA 3'-hinge interaction with region E1 of the external transcribed spacer (ETS) of pre-rRNA. This interaction is crucial for docking to initiate rRNA processing. 18S rRNA production was inhibited when fewer than 6 of the 8 bp of the U3 3'-hinge complex with the ETS could form; moreover, base pairing involving the right side of the 3'-hinge was more important than the left. Increasing the length of the U3 hinge-ETS interaction by 9 bp impaired rRNA processing. Formation of 18S rRNA was also inhibited by swapping the U3 5'- and 3'-hinge interactions with the ETS or by shifting the base pairing of the U3 3'-hinge to the sequence directly adjacent to ETS region E1. However, 18S rRNA production was partially restored by a compensatory shift that allowed the sequence adjacent to the U3 3'-hinge to pair with the eight bases directly adjacent to ETS region E1. The results suggest that the geometry of the U3 snoRNA interaction with the ETS is critical for rRNA processing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gki815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1199564PMC
September 2005

Complex mosaicism is a novel approach to infectivity enhancement of adenovirus type 5-based vectors.

Cancer Gene Ther 2005 May;12(5):475-86

VectorLogics Inc., 550 South 11th Street, CRC-122A, Birmingham, AL 35294, USA.

The use of adenovirus type 5 (Ad5) for cancer therapy is limited by deficiency of its primary cell attachment receptor, coxsackie and adenovirus receptor (CAR), on cancer cells. Ad5 retargeting to alternate receptors through fiber genetic modification can be used to circumvent CAR dependence of its tropism, and thereby achieve infectivity enhancement. Here we propose and test a novel "complex mosaicism" approach for fiber modification, which combines serotype chimerism with peptide ligand(s) incorporation in a single-fiber molecule. We incorporated integrin-binding peptide RGD-4C in the HI-loop, at the carboxy (C)-terminus, or both locales of the Ad3 knob, in the context of Ad5/3 chimera fiber in order to retarget simultaneously the Ad vector to integrins and Ad3 receptors. The infectivity enhancement of the fiber modifications was assessed in various cancer cell lines as cancer-targeting models. Replication-defective complex mosaic Ad-luc vectors bearing chimeric fiber (F.5/3), with or without C-terminal RGD-modification of Ad3 knob, demonstrated up to 55-fold gene transfer increase in bladder cancer cell lines. Although this augmentation was primarily due to Ad3 receptor targeting, some contribution of RGD-mediated integrin-targeting was also observed, suggesting that complex mosaic modification can function in a dual-receptor targeting via a single Ad3 fiber knob.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.cgt.7700806DOI Listing
May 2005

Xenopus U3 snoRNA docks on pre-rRNA through a novel base-pairing interaction.

RNA 2004 Jun;10(6):942-53

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Division of Biology and Medicine, Providence, Rhode Island 02912, USA.

U3 small nucleolar RNA (snoRNA) is essential for rRNA processing to form 18S ribosomal RNA (rRNA). Previously, it has been shown that nucleolin is needed to load U3 snoRNA on pre-rRNA. However, as documented here, this is not sufficient. We present data that base-pairing between the U3 hinges and the external transcribed spacer (ETS) is critical for functional alignment of U3 on its pre-rRNA substrate. Additionally, the interaction between the U3 hinges and the ETS is proposed to serve as an anchor to hold U3 on the pre-rRNA substrate, while box A at the 5' end of U3 snoRNA swivels from ETS contacts to 18S rRNA contacts. Compensatory base changes revealed base-pairing between the 3' hinge of U3 snoRNA and region E1 of the ETS in Xenopus pre-rRNA; this novel interaction is required for 18S rRNA production. In contrast, base-pairing between the 5' hinge of U3 snoRNA and region E2 of the ETS is auxiliary, unlike the case in yeast where it is required. Thus, higher and lower eukaryotes use different interactions for functional association of U3 with pre-rRNA. The U3 hinge sequence varies between species, but covariation in the ETS retains complementarity. This species-specific U3-pre-rRNA interaction offers a potential target for a new class of antibiotics to prevent ribosome biogenesis in eukaryotic pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1370586PMC
http://dx.doi.org/10.1261/rna.5256704DOI Listing
June 2004

U4 snRNA nucleolar localization requires the NHPX/15.5-kD protein binding site but not Sm protein or U6 snRNA association.

J Cell Biol 2003 Sep 25;162(5):821-32. Epub 2003 Aug 25.

Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.

All small nuclear RNAs (snRNAs) of the [U4/U6.U5] tri-snRNP localize transiently to nucleoli, as visualized by microscopy after injection of fluorescein-labeled transcripts into Xenopus laevis oocyte nuclei. Here, we demonstrate that these RNAs traffic to nucleoli independently of one another, because U4 snRNA deleted in the U6 base-pairing region still localizes to nucleoli. Furthermore, depletion of endogenous U6 snRNA does not affect nucleolar localization of injected U4 or U5. The wild-type U4 transcripts used here are functional: they exhibit normal nucleocytoplasmic traffic, associate with Sm proteins, form the [U4/U6] di-snRNP, and localize to nucleoli and Cajal bodies. The nucleolar localization element (NoLE) of U4 snRNA was mapped by mutagenesis. Neither the 5'-cap nor the 3'-region of U4, which includes the Sm protein binding site, are essential for nucleolar localization. The only region in U4 snRNA required for nucleolar localization is the 5'-proximal stem loop, which contains the binding site for the NHPX/15.5-kD protein. Even mutation of just five nucleotides, essential for binding this protein, impaired U4 nucleolar localization. Intriguingly, the NHPX/15.5-kD protein also binds the nucleolar localization element of box C/D small nucleolar RNAs, suggesting that this protein might mediate nucleolar localization of several small RNAs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200301071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172826PMC
September 2003

The nucleolus: a site of ribonucleoprotein maturation.

Curr Opin Cell Biol 2003 Jun;15(3):318-25

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Division of Biology and Medicine, JW Wilson Laboratory, 69 Brown Street, Providence, RI 02912, USA.

The nucleolus is the site of ribosomal RNA synthesis, processing and ribosome maturation. Various small ribonucleoproteins also undergo maturation in the nucleolus, involving RNA modification and RNA-protein assembly. Such steps and other activities of small ribonucleoproteins also take place in Cajal (coiled) bodies. Events of ribosome biogenesis are found solely in the nucleolus, which is the final destination of small nucleolar RNAs after their traffic through Cajal bodies. However, nucleoli are just a stopping point in the intricate cellular traffic for small nuclear RNAs and other ribonucleoproteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0955-0674(03)00049-8DOI Listing
June 2003
-->