Dr.Indulekha Pillai, PhD

Our current research focusses on understanding the molecular mechanisms of cardiovascular diseases(CVDs), that are the leading cause of mortality and morbidity globally. We are working at the interface of interdisciplinary domains, stem cell biology, immunology, and bioengineering to produce novel therapeutic strategies against CVDs. Two primary research directions of the group are: 

 

• Immune Regulation of Cardiac regeneration

 

Injury in the adult heart leads to the loss of functional cardiomyocytes (the beating cells). The loss of cells triggers an immune response, which regulates the regenerative response. As human heart doesn’t have the robust regenerative ability, and thus heals primarily by forming non-functional scar tissue (fibrosis). We understand how immune response following an injury modulates regeneration (formation of new functional cardiomyocytes) vs. fibrosis.

 

• Molecular regulators of cardiac calcification

 

Aging, damage, and metabolic diseases can lead to calcification in the cardiovascular system. Calcification is the abnormal deposition of a bony matrix in soft tissue that can cause a mechanical or electrical block in the conduction system of the heart. The blockage can disrupt the smooth propagation of electric impulses leading to arrhythmias. 

Calcification is an active cell-mediated process where bone-forming cells (osteoblast progenitors), mature and differentiate to induce mineralization. We have recently shown that cardiac fibroblasts in the adult heart adopt an osteoblast-like fate and induce mineralization in the heart (Pillai et al Cell Stem Cell, 2017). Currently, there are no treatment modalities available to treat cardiac calcification. We are investigating the molecular regulation of cardiovascular calcification to develop better therapeutic options. 

Thus using stem cells, bio-engineering principles, and high-resolution imaging techniques, we are exploring the molecular mechanisms modulating regeneration, fibrosis, and calcification in the heart. Our ultimate goal is to augment the regeneration of the adult heart to produce a better functional outcome.

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My major contributions during my post-doctoral and doctoral fellowships are:

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1. Demonstrated the progenitor role of adult cardiac fibroblast in calcification and neovascularisation during cardiac injury

 

Cardiac calcification is one of the major complications associated with chronic kidney disease, aging etc. which cause heart conduction blocks and heart failure eventually. By genetic lineage tracing, using mice having genetically labelled cardiac fibroblasts in multiple mouse models of cardiac injury, I demonstrated for the first time that cardiac fibroblast adopt a progenitor like cell fate and induce mineralisation (Indulekha CL Pillai et al Cell Stem Cell online, November 14,2016). Cardiac fibroblast upon injury, express osteo-progenitor markers and secrete extracellular matrix protein. Moreover, using in vivo transplantation of cardiac fibroblasts in ectopic location, I demonstrated that cardiac fibroblasts are able to induce mineralization directly. Further, I found that the calcification process in heart is mediated by ENPP1, an enzyme that gets activated in cardiac fibroblasts upon injury. Small molecules of ENNP1 or inhibitors of bone mineralization completed prevented calcification with improved cardiac function after injury. Thus in summary, I demonstrated the progenitor role of cardiac fibroblast in calcification using in-vivo and in-vitro models of calcification and provided pharmacological agents for therapeutic development. Manuscript out of this work is published in Cell Stem Cell (Indulekha et al Cell Stem Cell 2017) and was highlighted by Science Magazine (Editor’s choice December 2016). Also the work is selected as the cover story of Cell Stem cell, February 2017 issue.

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2. Extracellular Niche in progenitor activation and subsequent differentiation in acute epileptic injury.

 

In this project, I demonstrated that in homeostatic conditions of brain, progenitors are present in a quiescent state and gets activated in response to injury. To demonstrate the progenitor signature in adult hippocampus after injury, I used pilocarpine induced rat injury model, a widely used animal model of temporal lobe epilepsy. After induction of epilepsy, hippocampal tissues were collected at different time points after injury along with uninjured controls. Using immunohistochemical and RT PCR analysis of different neural progenitor markers, I found that neural progenitors gets activated immediately after injury in hippocampus. Subsequently by BrdU incorporation assays, I found that the activated progenitors undergo rapid proliferation by 3 days of injury. Within 1 to 2 weeks, few activated progenitors differentiated into immature neurons only in the dentate gyrus region of hippocampus. However I found that inter cellular connections made by this new neurons were not normal. In parallel, quantification of growth factors in hippocampus, revealed that expression of growth factors such as FGF2, EGF, CNTF and BDNF were increased in the hippocampus during the course of neural progenitor division and maturation. This suggests a profound effect of micro environmental niche in stimulation of adult neural progenitors ().

Functional role of growth factors in neural progenitor proliferation and differentiation were again confirmed using embryonic stem cell (ES) derived neuronal differentiation strategies. We differentiated ES cell derived neural progenitors in presence and absence of growth factors such as EGF and FGF2 with appropriate controls and studied their effect of those factors in proliferation and differentiation of neural progenitors. We demonstrated that addition of EGF leads to more glial progenitors and subsequently more glial cells. However, treatment of FGF2 induces more neural progenitors and thus more neurons. Thus we observed by immunohistochemical analysis and by RT PCR that treating with FGF2 leads to more neurons and treating with EGF leads to more glial cells at the expense of neurons. So treatment of FGF2 primes them to a neuronal fate and EGF primes them to a glial fate (Sanal Kumar 2010).

Further I studied the effect of FGF2 and Shh (sonic hedgehog) in retinal ganglion cell differentiation in collaboration with other graduate students in the lab. For that we differentiated ES cell derived neural progenitors into neurons and exposed the progenitors with either Shh or FGF2 alone or in combination. We found that Shh in combination with FGF2 leads to increased differentiation to retinal ganglion cells (RGCs). RGCs are cells that have been irreversibly damaged in glaucoma, one of the leading cause of blindness (Jagatha 2009 BBRC).

1. Seizure induces activation of multiple subtypes of neural progenitors and growth factors in hippocampus with neuronal maturation confined to dentate gyrus. CL Indulekha, Rajendran Sanalkumar, Anoop K Thekkuveettil & Jackson James. Biochem Biophys Res Commun. Volume 393, Issue 4, 19 March 2010, Pages 864-871.

2. Neuronal Vs. glial fate of embryonic stem cell derived neural progenitors (ES-NPs) are determined by FGF2/EGF during proliferation. R Sanalkumar, S Vidyanand, C L Indulekha, & Jackson James. Journal of Molecular Neuroscience, September 2010, Volume 42, Issue 1, pp 17-27

3. In vitro differentiation of retinal ganglion-like cells from embryonic stem cell derived neural progenitors. B Jagatha, MS Divya, R Sanalkumar, C L. Indulekha, S Vidyanand, TS. Divya, Ani V. Das & Jackson James. Biochemical and Biophysical Research Communications 380 (2009) 230–235

 

3.Role of Notch signalling in progenitor proliferation and differentiation

 

Neural progenitors can either be differentiated into neurons or glial cells. Moreover the newly formed neurons can either be excitatory or inhibitory. Excitatory neurons carry mostly glutamate as the neurotransmitter and inhibitory neurons use GABA as the neurotransmitter. Tlx3 is a selector gene that regulates the differentiation of glutamatergic Vs GABAergic differentiation. In order to elucidate the molecular regulators of neuronal subtype specification, I studied the molecular regulation of Tlx3 gene. For that, I cloned the upstream promoter region and using promoter reporter assays, RT PCR and DNA protein interaction studies; I demonstrated the role of notch signaling in neuronal subtype specification. I found that a Notch target gene, Hes1, directly binds to Tlx3 promoter and drives the neuron to a GABAergic fate.  Moreover over expression of Hes1 in ES cell derived neural progenitor’s leads to increased GABAergic differentiation whereas down regulation of Hes1 using dominant negative Hes1 leads to more glutamatergic neurons.

 We studied the role of Notch signaling in neural progenitor proliferation and maintenance in addition to its role in differentiation. We found that ATF2 binds to Hes1 promoter and regulates the expression of Hes1 and helps in maintenance of progenitors. Using notch and Hes1 promoter reporter assay, western blotting, immune cytochemistry, we demonstrated that ATF2 maintain a subset of neural progenitors through CBF1/Notch independent Hes-1 expression and synergistically activates the expression of Hes-1 in Notch dependent neural progenitors.

1. Hes-1 regulates the excitatory fate of neural progenitors through modulation of Tlx3 (HOX11L2) expression. Indulekha CL, Divya TS, Divya MS, Sanalkumar R, Rasheed VA, Dhanesh SB, Sebin A, George A, James J. Cellular and Molecular Life Sciences, February 2012, Volume 69, Issue 4, pp 611-627

2. ATF2 maintains a subset of neural progenitors through CBF1/Notch independent Hes-1 expression and synergistically activates the expression of Hes-1 in Notch dependent neural progenitors. R Sanalkumar, CL Indulekha, T S Divya, M S Divya, R J Anto, B Vinod, S Vidyanand, B Jagatha, S Venugopal & J James.     Journal of Neurochemistry, Volume 113, Issue 4, pages 807–818, May 2011.

 

4.Role of RanBP2, in cone photoreceptor development

 

I contributed towards understanding the role of Ran binding protein-2, a nuclear protein using cell type specific knockdown based on Cre-lox P recombination techniques. I found that Ran binding protein-2 is required for the proper maintenance of cone photoreceptor, and its knockdown leads to degeneration of cells through apoptosis.

1. Probing the role(s) of the loss of RAN-Binding Protein-2 (RANBP2) in M-cone photoreceptors and sub-populations of brain neurons. Indulekha Chandrasekharan Pillai Lalitha, Nomingerel Tserentsoodol, Kyoung-in Cho, Paulo A Ferreira. Investigative Ophthalmology & Visual Science (2011) 52 (14), 1816-1816.

2. Distinct and atypical intrinsic and extrinsic cell death pathways between photoreceptor cell types upon specific ablation of Ranbp2 in cone photoreceptors, Cho K., Haque, M., Wang, J, Yu, M., Hao, Y., Qiu, S., Indulekha, CL Pillai., Peachey, N. and Ferreira, PA (2013) PloS Genetics, 9(6): e1003555 (PMCID: PMC3688534)

 

5.Role of P65, an NF ƘB subunit in Type II alveolar cell mediated repair process in response to bleomycin induced lung injury.

 

During my tenure in Cedars Sinai Medical Centre Los Angeles, I studied the role of p65, an NF KappaB subunit in TypeII alveolar cell renewal and differentiation. Preliminary studies in p65 cell specific knock out mice, shows absence of p65 leads to reduced proliferation of TypeII alveolar cells. Self-renewal of alveolar type II cells is essential for the lung injury mediated repair process.

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