Publications by authors named "Harmandeep Kaur"

21 Publications

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1 E-Cadherin knockdown induces cancer stem cell-like phenotype and drug resistance.

Biochem Cell Biol 2021 Mar 6. Epub 2021 Mar 6.

Post Graduate Institute of Medical Education and Research, 29751, Biophysics, Chandigarh, India, 160012;

Cervical cancer is one of the leading causes of mortality amongst women in developing countries and therapy resistance is the main reason for its treatment failure. Recent advances suggest that cancer stem cells (CSCs) are critically involved in regulating the chemo resistant behavior of cervical cancer cells. In our study the CSC phenotype cells were isolated and the expression of stem cell marker and epithelial-mesenchymal transition (EMT) associated gene was confirmed by various assays. However, these CSC phenotype cells cannot be cultured for further cytotoxicity studies. So, we tried to establish a CSC model in cervical cancer cells. We performed the siRNA-mediated knockdown of E-cadherin (E-cad) in these cells and studied EMT associated stem cell-like properties in them. We also performed dose dependent cell viability assay using clinically relevant drugs such as cisplatin, cyclopamine and GANT58 to analyze the drug resistant behavior of these cancer cells. We found that E-cad knockdown induces EMT in cervical cancer cells imparting stem-cell like characteristics along with enhanced tumorsphere formation, migration, invasion ability and drug resistance. This is the first study to establish a CSC model in cervical cancer cells by knockdown of E-cad which can be utilized for development of anti-cancer therapies.
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http://dx.doi.org/10.1139/bcb-2020-0592DOI Listing
March 2021

Effect of Fentanyl on Block Characteristics as Adjuvant to Intrathecal Bupivacaine for Lower Limb Surgeries.

Anesth Essays Res 2020 Apr-Jun;14(2):343-348. Epub 2020 Oct 12.

Department of Anaesthesia, SGRDIMSAR, Amritsar, Punjab, India.

Background: Regional anesthesia is the preferred technique for most of lower abdominal and lower limb surgeries as it allows the patient to remain awake and minimize the problems associated with airway management. Hyperbaric bupivacaine 0.5%, although extensively used for spinal anesthesia, has a limitation of short duration. The addition of fentanyl, a synthetic lipophilic opioid, is known to prolong postoperative analgesia.

Aims: We aimed to study the effect of the addition of different doses of fentanyl to hyperbaric bupivacaine about hemodynamic changes, the extent of sensory and motor block, duration of analgesia, and complications that occur during the procedure.

Settings And Design: This study was a prospective, comparative, randomized, and double-blind study.

Materials And Methods: Patients were randomly allocated to three groups of 30 each. Group I (n [number of patients] = 30) received bupivacaine 0.5% heavy 2.0 mL diluted up to 2.5 mL with normal saline. Group II ( = 30) received bupivacaine 0.5% heavy 2.0 mL and fentanyl 20 μg diluted up to 2.5 mL with normal saline, and Group III ( = 30) received bupivacaine 0.5% heavy 2.0 mL and fentanyl 50 μg diluted up to 2.5 mL with normal saline.

Statistical Analysis: The data were analyzed using Chi-square and Student's -test.

Results And Conclusions: The onset of sensory and motor block was early in Group III in comparison to Group I and Group II ( < 0.05). The duration of analgesia was significantly longer in Group III, followed by Group II, and least in Group I. None of the patients in Groups I and II had any complications such as hypotension, nausea, vomiting, bradycardia, and pruritus. However, the incidence of hypotension, nausea, and pruritus was more in Group III. 2 mg intrathecal bupivacaine with 20 μg fentanyl provides reliable and satisfactory sensory and motor block without increasing the incidence of side effects.
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http://dx.doi.org/10.4103/aer.AER_58_20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819414PMC
October 2020

The study of single cells in diabetic kidney disease.

J Nephrol 2021 Jan 21. Epub 2021 Jan 21.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael's Hospital, 6-151 61 Queen Street East, Toronto, ON, M5C 2T2, Canada.

In the past few years there has been a rapid expansion of interest in the study of single cells, especially through the new techniques that involve single-cell RNA sequencing (scRNA-seq). Recently, these techniques have provided new insights into kidney health and disease, including insights into diabetic kidney disease (DKD). However, despite the interest and the technological advances, the study of individual cells in DKD is not a new concept. Many clinicians and researchers who work within the DKD space may be familiar with experimental techniques that actually involve the study of individual cells, but may be unfamiliar with newer scRNA-seq technology. Here, with the goal of improving accessibility to the single-cell field, we provide a primer on single-cell studies with a focus on DKD. We situate the technology in its historical context and provide a brief explanation of the common aspects of the different technologies available. Then we review some of the most important recent studies of kidney (patho)biology that have taken advantage of scRNA-seq techniques, before emphasizing the new insights into the molecular pathogenesis of DKD gleaned with these techniques. Finally, we highlight common pitfalls and limitations of scRNA-seq methods and we look toward the future to how single-cell experiments may be incorporated into the study of DKD and how to interpret the findings of these experiments.
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http://dx.doi.org/10.1007/s40620-020-00964-1DOI Listing
January 2021

Biamphiphilic ionic liquid based aqueous microemulsions as an efficient catalytic medium for cytochrome .

Phys Chem Chem Phys 2021 Jan;23(1):320-328

Department of Chemistry, UGC Sponsored Centre for Advanced Studies-II, Guru Nanak Dev University, Amritsar-143005, India.

Considering the remarkable applicability of ionic liquids (ILs) in bio-catalysis involving enzymes, herein, we report new IL based aqueous microemulsions as a catalytic reactor for cytochrome c (Cyt-c). Microemulsions (μEs), comprising water as the polar component, imidazolium (cation) and dioctylsulfosuccinate (AOT) (anion) based biamphiphilic ionic liquid (BAIL) as the surfactant and a hydrophobic ionic liquid (HIL) as the non-polar component have been prepared and characterized. The use of BAIL has promoted the formation of μEs without any co-surfactant, owing to its higher surface activity. The effect of ester- or amide-functionalization of the alkyl chain of the imidazolium cation of BAILs on the phase behavior of μEs has been investigated. The prepared μEs have been characterized via conductivity, dynamic light scattering (DLS), UV-vis absorption and steady-state fluorescence (using external polarity probes) techniques. The prepared μEs have been employed as nano-reactors for exploring the catalytic activity of Cyt-c. The formed BAIL-water nano-interfaces in reverse μEs have exerted a positive effect on the catalytic activity of Cyt-c stored in a water pool of reverse μEs. A five-fold higher rate constant in μEs as compared to buffer establishes μEs as a better catalytic medium. Furthermore, the differing nature of nano-interfaces created by BAILs and water in reverse μEs, depending on the functionalization of the alkyl chain of the cationic part of BAIL, has exerted varying influence on the catalytic activity of Cyt-c. It is expected that the present work will result in providing a versatile platform for the creation of new IL and water based μEs for bio-catalytic applications.
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http://dx.doi.org/10.1039/d0cp04513fDOI Listing
January 2021

Lung and Kidney ACE2 and TMPRSS2 in Renin-Angiotensin System Blocker-Treated Comorbid Diabetic Mice Mimicking Host Factors That Have Been Linked to Severe COVID-19.

Diabetes 2021 03 11;70(3):759-771. Epub 2020 Dec 11.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada

The causes of the increased risk of severe coronavirus disease 2019 (COVID-19) in people with diabetes are unclear. It has been speculated that renin-angiotensin system (RAS) blockers may promote COVID-19 by increasing ACE2, which severe acute respiratory syndrome coronavirus 2 uses to enter host cells, along with the host protease TMPRSS2. Taking a reverse translational approach and by combining in situ hybridization, primary cell isolation, immunoblotting, quantitative RT-PCR, and liquid chromatography-tandem mass spectrometry, we studied lung and kidney ACE2 and TMPRSS2 in diabetic mice mimicking host factors linked to severe COVID-19. In healthy young mice, neither the ACE inhibitor ramipril nor the AT1 receptor blocker telmisartan affected lung or kidney ACE2 or TMPRSS2, except for a small increase in kidney ACE2 protein with ramipril. In contrast, mice with comorbid diabetes (aging, high-fat diet, and streptozotocin-induced diabetes) had heightened lung ACE2 and TMPRSS2 protein levels and increased lung ACE2 activity. None of these parameters were affected by RAS blockade. ACE2 was similarly upregulated in the kidneys of mice with comorbid diabetes compared with aged controls, whereas TMPRSS2 (primarily distal nephron) was highest in telmisartan-treated animals. Upregulation of lung ACE2 activity in comorbid diabetes may contribute to an increased risk of severe COVID-19. This upregulation is driven by comorbidity and not by RAS blockade.
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http://dx.doi.org/10.2337/db20-0765DOI Listing
March 2021

To compare the analgesic efficacy of two different doses of epidural ketamine in chronic low back-pain patients: A randomised double-blind study.

Indian J Anaesth 2020 Sep 1;64(9):768-773. Epub 2020 Sep 1.

Department of Anaesthesia, Sri Guru Ram Das Institute of Medical Science and Research, Sri Amritsar, Punjab, India.

Background And Aims: Ketamine, an adjunct to epidural steroid injections (ESI) for chronic back-pain provides better quality and prolonged duration of analgesia. The present study aims to evaluate the analgesic efficacy in terms of pain scores, duration of pain-free period, patient satisfaction score (PSS) and number of repeat injections with 25 mg versus 50 mg ketamine as adjuvants to ESI.

Methods: In a prospective, randomised, double-blind trial at a tertiary care hospital, 60 patients of chronic low back-pain of either sex, aged 18-65 years, received preservative free 25 mg ketamine in Group I and 50 mg ketamine in Group II as adjunct to 40 mg triamcinolone in total 6 ml volume given epidurally. Baseline data along with follow-ups at 2, 4, 8 and 12 weeks post-procedure included assessment of pain using Visual Analogue Scale (VAS), duration, number of repeat blocks using PSS, Quality of Life (QoL) and side-effects. Categorical data analysed using the Chi-Square test, and continuous data using paired -test.

Results: Pain evaluation within the groups over time showed significant improvement from baseline ( = 0.000), and between the groups showed comparable VAS scores at 12 weeks ( = 0.392). The PSS, pain-free duration and number of repeat injections were also statistically comparable. However, the QoL improved more in Group II vs Group I ( = 0.024). The short-lasting side effects were more in Group II, but no features of neurotoxicity were observed in any patient.

Conclusion: The analgesic efficacy of adjuvant therapy with 50 mg ketamine appeared comparable to 25 mg ketamine. Although, there was a better quality of life and longer pain-free interval with 50 mg ketamine, the side effects were more.
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http://dx.doi.org/10.4103/ija.IJA_541_20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641091PMC
September 2020

Cell-free miRNAs as non-invasive biomarkers in breast cancer: Significance in early diagnosis and metastasis prediction.

Life Sci 2020 Apr 7;246:117417. Epub 2020 Feb 7.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada. Electronic address:

Breast cancer is one of the genetic diseases causing a high mortality among women around the world. Despite the availability of advanced diagnostic tools and treatment strategies, the incidence of breast cancer is increasing every year. This is due to the lack of accurate and reliable biomarkers whose deficiency creates difficulty in early breast cancer recognition, subtypes determination, and metastasis prophecy. Although biomarkers such as ER, PR, Her2, Ki-67, and other genetic platforms e.g. MammaPrint®, Oncotype DX®, Prosigna® or EndoPredict® are available for determination of breast cancer diagnosis and prognosis. However, pertaining to heterogeneous nature, lack of sensitivity, and specificity of these markers, it is still incessant to overcome breast cancer burden. Therefore, a novel biomarker is urgently needed for therapeutic diagnosis and improving prognosis. Lately, it has become more evident that cell-free miRNAs might be useful as good non-invasive biomarkers that are associated with different events in carcinogenesis. For example, some known biomarkers such as miR-21, miR-23a, miR-34a are associated with molecular subtyping and different biomolecular aspects i.e. apoptosis, angiogenesis, metastasis, and miR-1, miR-10b, miR-16 are associated with drug response. Cell-free miRNAs present in human body fluids have proven to be potential biomarkers with significant prognostic and predictive values. Numerous studies have found a distinct expression profile of circulating miRNAs in breast tumour versus non-tumour and in early and advanced-stage, thus implicating its clinical relevance. This review article will highlight the importance of different cell-free miRNAs as a biomarker for early breast cancer detection, subtype classification, and metastasis forecast.
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http://dx.doi.org/10.1016/j.lfs.2020.117417DOI Listing
April 2020

Orphan G Protein-Coupled Receptor GPRC5B Controls Smooth Muscle Contractility and Differentiation by Inhibiting Prostacyclin Receptor Signaling.

Circulation 2020 04 16;141(14):1168-1183. Epub 2020 Jan 16.

Department of Pharmacology (J.C., R.C., R.L., H.K., W.Z., S.T., S.O., N.W.), Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

Background: G protein-coupled receptors are important regulators of contractility and differentiation in vascular smooth muscle cells (SMCs), but the specific function of SMC-expressed orphan G protein-coupled receptor class C group 5 member B (GPRC5B) is unclear.

Methods: We studied the role of GPRC5B in the regulation of contractility and dedifferentiation in human and murine SMCs in vitro and in iSM--KO (tamoxifen-inducible, SMC-specific knockout) mice under conditions of arterial hypertension and atherosclerosis in vivo.

Results: Mesenteric arteries from SMC-specific -KOs showed ex vivo significantly enhanced prostacyclin receptor (IP)-dependent relaxation, whereas responses to other relaxant or contractile factors were normal. In vitro, knockdown of GPRC5B in human aortic SMCs resulted in increased IP-dependent cAMP production and consecutive facilitation of SMC relaxation. In line with this facilitation of IP-mediated relaxation, iSM--KO mice were protected from arterial hypertension, and this protective effect was abrogated by IP antagonists. Mechanistically, we show that knockdown of GPRC5B increased the membrane localization of IP both in vitro and in vivo and that GPRC5B, but not other G protein-coupled receptors, physically interacts with IP. Last, we show that enhanced IP signaling in GPRC5B-deficient SMCs not only facilitates relaxation but also prevents dedifferentiation during atherosclerosis development, resulting in reduced plaque load and increased differentiation of SMCs in the fibrous cap.

Conclusions: Taken together, our data show that GPRC5B regulates vascular SMC tone and differentiation by negatively regulating IP signaling.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043703DOI Listing
April 2020

Assessment of shelf-life of extract through WHO recommended stability study involving chromatographic and biological activity analyses.

Nat Prod Res 2019 Oct 29:1-6. Epub 2019 Oct 29.

Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, India.

Stability study on extract under WHO recommended accelerated and long-term conditions for 6 and 30 months, respectively was carried out by taking gymnemagenin as a marker and by evaluating antidiabetic activity through different models. Gymnemagenin was not detected in any stability sample indicating that gymnemic acids (GAs) remain stable in the extract under the test conditions. The extract and its GA rich fraction exhibited mild α-glucosidase inhibitory activity (18-27%) that remained same during the study. Neither hypoglycemic nor anti-hyperglycemic effect was induced by the extract in normal rats in oral glucose tolerance test. The extract and GA rich fraction showed significant antidiabetic activity in alloxan-induced diabetic rats that remained same in all stability samples. Based on these findings, a shelf-life of at least 30 months is suggested for extract under long-term conditions, and gymnemagenin as a marker for shelf-life assessment of products derived from the plant.
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http://dx.doi.org/10.1080/14786419.2019.1682578DOI Listing
October 2019

Comparative Study of Effectiveness of Tramadol and Butorphanol as Adjuvants to Levobupivacaine for Supraclavicular Brachial Plexus Block.

Anesth Essays Res 2019 Jul-Sep;13(3):446-451

Department of Anaesthesia, Sri Guru Ram Das Institute of Medical Sciences and Research, Sri Amritsar, Punjab, India.

Background: Butorphanol and tramadol, the synthetic opioid analgesics, have been used alone or in combination with a local anesthetic in supraclavicular brachial plexus block.

Aim: The aim of this study was to evaluate the sensory and motor block characteristics with the addition of tramadol (100 mg) and butorphanol (2 mg) to levobupivacaine for supraclavicular brachial plexus anesthesia.

Settings And Design: This study was a prospective, randomized, double-blind, and comparative study.

Materials And Methods: Patients were randomly allocated to three groups of 30 each. Group A received 0.5% levobupivacaine (25 mL) and saline in 5 mL, Group B received 0.5% levobupivacaine in 30 mL with 100 mg tramadol, and Group C received 0.5% levobupivacaine in 30 mL with 2 mg butorphanol. The duration of sensory block was evaluated as a primary outcome and other parameters as secondary outcomes.

Statistical Tests: Statistical analyses were performed using Chi-square test for nonparametric data and analysis of variance for parametric data.

Results: Onset time of sensory block was fast and comparable in both Group B and C as compared to Group A ( = 0.000). Group C had the longest duration of sensory block and duration of analgesia followed by Group B and Group A. Group C and Group B showed statistically longer duration of motor block as compared to Group A ( = 0.000). Hemodynamic derangements and adverse effects were comparable.

Conclusion: Butorphanol (2 mg) as an adjuvant to levobupivacaine in supraclavicular block hastens the onset and prolongs the duration of the block as well as postoperative analgesia to a greater extent as compared to the addition of 100 mg tramadol.
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http://dx.doi.org/10.4103/aer.AER_110_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775833PMC
October 2019

Single-cell profiling reveals GPCR heterogeneity and functional patterning during neuroinflammation.

JCI Insight 2017 Aug 3;2(15). Epub 2017 Aug 3.

Department of Pharmacology.

GPCR expression was intensively studied in bulk cDNA of leukocyte populations, but limited data are available with respect to expression in individual cells. Here, we show a microfluidic-based single-cell GPCR expression analysis in primary T cells, myeloid cells, and endothelial cells under naive conditions and during experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis. We found that neuroinflammation induces characteristic changes in GPCR heterogeneity and patterning, and we identify various functionally relevant subgroups with specific GPCR profiles among spinal cord-infiltrating CD4 T cells, macrophages, microglia, or endothelial cells. Using GPCRs CXCR4, S1P1, and LPHN2 as examples, we show how this information can be used to develop new strategies for the functional modulation of Th17 cells and activated endothelial cells. Taken together, single-cell GPCR expression analysis identifies functionally relevant subpopulations with specific GPCR repertoires and provides a basis for the development of new therapeutic strategies in immune disorders.
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http://dx.doi.org/10.1172/jci.insight.95063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543912PMC
August 2017

The zebrafish ventricle: A hub of cardiac endothelial cells for in vitro cell behavior studies.

Sci Rep 2017 06 2;7(1):2687. Epub 2017 Jun 2.

Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany.

Despite our increasing understanding of zebrafish heart development and regeneration, there is limited information about the distribution of endothelial cells (ECs) in the adult zebrafish heart. Here, we investigate and compare the distribution of cardiac ECs (cECs) in adult mouse and zebrafish ventricles. Surprisingly, we find that (i) active coronary vessel growth is present in adult zebrafish, (ii) ~37 and ~39% of cells in the zebrafish heart are ECs and cardiomyocytes, respectively, a composition similar to that seen in mouse. However, we find that in zebrafish, ~36% of the ventricular tissue is covered with ECs, i.e., a substantially larger proportion than in mouse. Capitalising on the high abundance of cECs in zebrafish, we established a protocol to isolate them with high purity using fluorescent transgenic lines. Our approach eliminates side-effects due to antibody utilisation. Moreover, the isolated cECs maintained a high proliferation index even after three passages and were amenable to pharmacological treatments to study cEC migration in vitro. Such primary cultures will be a useful tool for supplementary in vitro studies on the accumulating zebrafish mutant lines as well as the screening of small molecule libraries on cardiac specific endothelial cells.
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http://dx.doi.org/10.1038/s41598-017-02461-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457396PMC
June 2017

Corticotropin releasing hormone receptor 2 exacerbates chronic cardiac dysfunction.

J Exp Med 2017 Jul 26;214(7):1877-1888. Epub 2017 May 26.

Department of Cardiology, Nagoya University School of Medicine, Nagoya, Japan.

Heart failure occurs when the heart is unable to effectively pump blood and maintain tissue perfusion. Despite numerous therapeutic advancements over previous decades, the prognosis of patients with chronic heart failure remains poor, emphasizing the need to identify additional pathophysiological factors. Here, we show that corticotropin releasing hormone receptor 2 (Crhr2) is a G protein-coupled receptor highly expressed in cardiomyocytes and continuous infusion of the Crhr2 agonist, urocortin 2 (Ucn2), reduced left ventricular ejection fraction in mice. Moreover, plasma Ucn2 levels were 7.5-fold higher in patients with heart failure compared to those in healthy controls. Additionally, cardiomyocyte-specific deletion of Crhr2 protected mice from pressure overload-induced cardiac dysfunction. Mice treated with a Crhr2 antagonist lost maladaptive 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent signaling and did not develop heart failure in response to overload. Collectively, our results indicate that constitutive Crhr2 activation causes cardiac dysfunction and suggests that Crhr2 blockade is a promising therapeutic strategy for patients with chronic heart failure.
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http://dx.doi.org/10.1084/jem.20161924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502432PMC
July 2017

A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib.

J Cell Biol 2017 Jan 22;216(1):199-215. Epub 2016 Dec 22.

Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany

Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor βPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration.
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http://dx.doi.org/10.1083/jcb.201602002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223600PMC
January 2017

Radial glia regulate vascular patterning around the developing spinal cord.

Elife 2016 11 17;5. Epub 2016 Nov 17.

Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

Vascular networks surrounding individual organs are important for their development, maintenance, and function; however, how these networks are assembled remains poorly understood. Here we show that CNS progenitors, referred to as radial glia, modulate vascular patterning around the spinal cord by acting as negative regulators. We found that radial glia ablation in zebrafish embryos leads to excessive sprouting of the trunk vessels around the spinal cord, and exclusively those of venous identity. Mechanistically, we determined that radial glia control this process via the Vegf decoy receptor sFlt1: mutants exhibit the venous over-sprouting observed in radial glia-ablated larvae, and sFlt1 overexpression rescues it. Genetic mosaic analyses show that sFlt1 function in trunk endothelial cells can limit their over-sprouting. Together, our findings identify CNS-resident progenitors as critical angiogenic regulators that determine the precise patterning of the vasculature around the spinal cord, providing novel insights into vascular network formation around developing organs.
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http://dx.doi.org/10.7554/eLife.20253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123865PMC
November 2016

Endothelial cation channel PIEZO1 controls blood pressure by mediating flow-induced ATP release.

J Clin Invest 2016 12 31;126(12):4527-4536. Epub 2016 Oct 31.

Arterial blood pressure is controlled by vasodilatory factors such as nitric oxide (NO) that are released from the endothelium under the influence of fluid shear stress exerted by flowing blood. Flow-induced endothelial release of ATP and subsequent activation of Gq/G11-coupled purinergic P2Y2 receptors have been shown to mediate fluid shear stress-induced stimulation of NO formation. However, the mechanism by which fluid shear stress initiates these processes is unclear. Here, we have shown that the endothelial mechanosensitive cation channel PIEZO1 is required for flow-induced ATP release and subsequent P2Y2/Gq/G11-mediated activation of downstream signaling that results in phosphorylation and activation of AKT and endothelial NOS. We also demonstrated that PIEZO1-dependent ATP release is mediated in part by pannexin channels. The PIEZO1 activator Yoda1 mimicked the effect of fluid shear stress on endothelial cells and induced vasorelaxation in a PIEZO1-dependent manner. Furthermore, mice with induced endothelium-specific PIEZO1 deficiency lost the ability to induce NO formation and vasodilation in response to flow and consequently developed hypertension. Together, our data demonstrate that PIEZO1 is required for the regulation of NO formation, vascular tone, and blood pressure.
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http://dx.doi.org/10.1172/JCI87343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127677PMC
December 2016

Lineage tracing of cells involved in atherosclerosis.

Atherosclerosis 2016 08 11;251:445-453. Epub 2016 Jun 11.

Department of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; Medical Faculty, Goethe University Frankfurt, Germany.

Background And Aims: Despite the clinical importance of atherosclerosis, the origin of cells within atherosclerotic plaques is not fully understood. Due to the lack of a definitive lineage-tracing strategy, previous studies have provided controversial results about the origin of cells expressing smooth muscle and macrophage markers in atherosclerosis. We here aim to identify the origin of vascular smooth muscle (SM) cells and macrophages within atherosclerosis lesions.

Methods: We combined a genetic fate mapping approach with single cell expression analysis in a murine model of atherosclerosis.

Results: We found that 16% of CD68-positive plaque macrophage-like cells were derived from mature SM cells and not from myeloid sources, whereas 31% of αSMA-positive smooth muscle-like cells in plaques were not SM-derived. Further analysis at the single cell level showed that SM-derived CD68(+) cells expressed higher levels of inflammatory markers such as cyclooxygenase 2 (Ptgs2, p = 0.02), and vascular cell adhesion molecule (Vcam1, p = 0.05), as well as increased mRNA levels of genes related to matrix synthesis such as Col1a2 (p = 0.01) and Fn1 (p = 0.04), than non SM-derived CD68(+) cells.

Conclusions: These results demonstrate that smooth muscle cells within atherosclerotic lesions can switch to a macrophage-like phenotype characterized by higher expression of inflammatory and synthetic markers genes that may further contribute to plaque progression.
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http://dx.doi.org/10.1016/j.atherosclerosis.2016.06.012DOI Listing
August 2016

Targeted Ablation of Periostin-Expressing Activated Fibroblasts Prevents Adverse Cardiac Remodeling in Mice.

Circ Res 2016 06 2;118(12):1906-17. Epub 2016 May 2.

From the Department of Pharmacology (H.K., C.Y.N., J.C., S.O., N.W.), Bioinformatics Facility (J.B., M.L.), Nuclear Magnetic Resonance Imaging Facility (A.W.), and Mass Spectrometry Group (A.P., S.H.), Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan (M.T.); Department of Pediatrics, Indiana University School of Medicine, Indianapolis (S.J.C.); Department of Cardiology, Kerckhoff Heart and Thorax Center, Bad Nauheim, Germany (H.M., C.T.); and Medical Faculty, J.W. Goethe University Frankfurt, Frankfurt, Germany (S.O., N.W.).

Rationale: Activated cardiac fibroblasts (CF) are crucial players in the cardiac damage response; excess fibrosis, however, may result in myocardial stiffening and heart failure development. Inhibition of activated CF has been suggested as a therapeutic strategy in cardiac disease, but whether this truly improves cardiac function is unclear.

Objective: To study the effect of CF ablation on cardiac remodeling.

Methods And Results: We characterized subgroups of murine CF by single-cell expression analysis and identified periostin as the marker showing the highest correlation to an activated CF phenotype. We generated bacterial artificial chromosome-transgenic mice allowing tamoxifen-inducible Cre expression in periostin-positive cells as well as their diphtheria toxin-mediated ablation. In the healthy heart, periostin expression was restricted to valvular fibroblasts; ablation of this population did not affect cardiac function. After chronic angiotensin II exposure, ablation of activated CF resulted in significantly reduced cardiac fibrosis and improved cardiac function. After myocardial infarction, ablation of periostin-expressing CF resulted in reduced fibrosis without compromising scar stability, and cardiac function was significantly improved. Single-cell transcriptional analysis revealed reduced CF activation but increased expression of prohypertrophic factors in cardiac macrophages and cardiomyocytes, resulting in localized cardiomyocyte hypertrophy.

Conclusions: Modulation of the activated CF population is a promising approach to prevent adverse cardiac remodeling in response to angiotensin II and after myocardial infarction.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.308643DOI Listing
June 2016

P2Y₂ and Gq/G₁₁ control blood pressure by mediating endothelial mechanotransduction.

J Clin Invest 2015 Aug 13;125(8):3077-86. Epub 2015 Jul 13.

Elevated blood pressure is a key risk factor for developing cardiovascular diseases. Blood pressure is largely determined by vasodilatory mediators, such as nitric oxide (NO), that are released from the endothelium in response to fluid shear stress exerted by the flowing blood. Previous work has identified several mechanotransduction signaling processes that are involved in fluid shear stress-induced endothelial effects, but how fluid shear stress initiates the response is poorly understood. Here, we evaluated human and bovine endothelial cells and found that the purinergic receptor P2Y2 and the G proteins Gq/G11 mediate fluid shear stress-induced endothelial responses, including [Ca2+]i transients, activation of the endothelial NO synthase (eNOS), phosphorylation of PECAM-1 and VEGFR-2, as well as activation of SRC and AKT. In response to fluid shear stress, endothelial cells released ATP, which activates the purinergic P2Y2 receptor. Mice with induced endothelium-specific P2Y2 or Gq/G11 deficiency lacked flow-induced vasodilation and developed hypertension that was accompanied by reduced eNOS activation. Together, our data identify P2Y2 and Gq/G11 as a critical endothelial mechanosignaling pathway that is upstream of previously described mechanotransduction processes and demonstrate that P2Y2 and Gq/G11 are required for basal endothelial NO formation, vascular tone, and blood pressure.
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http://dx.doi.org/10.1172/JCI81067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563756PMC
August 2015

Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate.

Nat Commun 2015 Apr 7;6:6658. Epub 2015 Apr 7.

Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231 Bad Nauheim, Germany.

The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKβ-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ∼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKβ substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKβ on serine 298. We provide evidence that IKKβ-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo.
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http://dx.doi.org/10.1038/ncomms7658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396366PMC
April 2015

Studies on scavenger receptors under experimental hypercholesterolemia: modulation on selenium supplementation.

Biol Trace Elem Res 2011 Oct 18;143(1):310-9. Epub 2010 Sep 18.

Department of Biophysics, Panjab University, Chandigarh 160014, India.

Scavenger receptors (SR) are the cell surface proteins that can bind and internalize modified lipoproteins. Because ox-LDL seems to play a key role in foam cell formation during atherogenesis, SR may be critical for pathogenesis of atherosclerosis. The present study was aimed to study the effect of selenium (Se) supplementation on SR, i.e., SRB1 and CD36 under experimental hypercholesterolemia. Male Sprague Dawley rats were divided into three groups and fed on the control diet, high cholesterol diet (HCD), and HCD + Se, respectively, for a period of 4 months. Selenium and reactive oxygen species (ROS) levels were estimated in serum and liver respectively. mRNA expression using RT-PCR and protein expression using ELISA were analyzed for SRB1 and CD36 receptors. Selenium levels decreased whereas ROS levels increased under experimental hypercholesterolemic state. Selenium supplementation (1 ppm), however, diminished the HCD-induced ROS levels. Furthermore, the protein expression of SRB1 was significantly reduced in HCD group in comparison to the control group. On the other hand, HCD-induced increase in CD36 mRNA and protein expression decreased significantly on Se supplementation. In conclusion, CD36 receptors seem to play a pro-atherogenic role under hypercholesterolemic state. Selenium supplementation, in addition, might prove to be a therapeutically valuable approach in near future to limit the adverse effect of hypercholesterolemia.
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http://dx.doi.org/10.1007/s12011-010-8833-8DOI Listing
October 2011