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John P. Cooke, MD, PhD Research group

Vascular Regeneration, Transdifferentiation, and “Transflammation”

Currently our work is focused on vascular regeneration, nuclear reprogramming and transdifferentiation.  We have expertise in the generation and characterization of induced pluripotent stem cells (iPSCs). We were the first to recognize the importance of innate immune activation in nuclear reprogramming to pluripotency (Lee et al, Cell 2012, FIGURE-1).  This work revealed that activation of innate immune signaling causes global changes in epigenetic modifiers that increases the probability of an open chromatin configuration which is necessary for reprogramming.  We have termed this process “Transflammation”.  Transflammation is a process that increases epigenetic plasticity to permit phenotypic fluidity.  Activation of pattern recognition receptors (PRRs) by damage-associated molecular patterns (DAMPs) or pathogen associated molecular patterns (PAMPs) activates cell signaling pathways that lead to global changes in the expression and activity of epigenetic modifiers, so as to enhance epigenetic plasticity.  In this way, the cell can increase its active genetic repertoire to respond to the challenge of pathogens or tissue injury. 

-Activation of innate immune signaling
-Global changes in expression and activity of epigenetic modifiers
-Open chromatin configuration
-Fluidity of cell phenotype
Lee et al, Cell 2012; Cooke JP, Circulation 2013; Sayed et al Circulation 2015; Zhou et al, Cell Reports 2016
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We have expertise in differentiating iPSCs or embryonic stem cells to endothelial cells (ECs), and have characterized the lymphatic, venous and arterial subtypes that are derived from iPSCs (Rufaihah et al, ATVB 2012).  Notably, based on our Cell paper, we have now systematically manipulated innate immune signaling to facilitate the process of transdifferentiation of one somatic cell to another.  This has permitted us to develop a small molecule based methodology to induce human fibroblasts to transdifferentiate into endothelial cells (iECs; Sayed et al Circulation 2015).  We have comprehensively characterized the endothelial nature of these cells and have demonstrated their therapeutic benefit in a pre-clinical model.  It is well known that inducible nitric oxide synthase (iNOS) is a major effector of innate immune signaling.  Activation of innate immunity leads to NFκB-mediated expression of iNOS, which S-nitrosates proteins to affect their activity and cellular location. Accordingly, we hypothesized that innate immune activation of iNOS, and S-nitrosation of epigenetic modifiers, might contribute to epigenetic plasticity required for transdifferentiation. Driven by this hypothesis, we are now studying novel iNOS-dependent post-translational modification of the repressive epigenetic machinery that facilitates transdifferentiation (Meng et al, Circulation Research 2016).   Endothelial functions are impaired in diabetes mellitus.  Most recently we find that hyperglycemia impairs the generation of iEC.  This intriguing finding may have relevance to diabetes-related vascular disease.

Endothelial Senescence

Endothelial senescence leads to vascular diseases, including atherosclerosis which causes heart attack and stroke.  We have found that a commonly used drug accelerates endothelial aging.  The proton pump inhibitors (PPIs) are commonly used for heartburn (gastroesophageal reflux).  They effectively block the gastric proton pump, reducing stomach acidity.   Unfortunately, we have found that the PPIs also block a lysosomal proton pump in endothelial cells.  Furthermore, at clinically relevant concentrations, the PPIs impair lysosomal acidity, reduce lysosomal enzyme activity, and cause the accumulation of protein aggregates in the endothelial cells, accelerating endothelial senescence(Yepuri et al, Circulation Research 2016; FIGURE-2).  This observation may explain the increased risk of heart attack, dementia and renal failure in PPI users.  Prolonged use of PPIs, such as Esomeprazole (Nexium) and omeprazole (Prilosec), has been associated with the development of several serious conditions, including chronic kidney disease, severe hypomagnesemia, infections, dementia, and increased incidence of cardiovascular events.  These drugs were never approved for longterm use, but are now over-the-counter and being used without medical supervision.  We intend to continue our investigations in animal models and humans, to determine the risk of long-term use of these drugs.

What follows is a description of a few of the therapeutic areas within cardiovascular regeneration that we are pursuing.  The therapeutic areas are arranged from those in the stage of discovery; those in pre-clinical development; to those ready for clinical studies.

Proton Pump Inhibitors (PPIs), common drugs for heartburn, impair lysosomal acidification and proteostasis of human endothelial cells. Protein aggregates accumulate, and daccelerate aging of endothelium. 

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Therapeutic areas-Discovery phase

Innate immunity in nuclear reprogramming

We have discovered that activation of innate immunity is critical in the nuclear reprogramming process that permits a cell to de-differentiate (e.g., into a pluripotent stem cell) or to transdifferentiate (into another somatic cell type).   In the November 2012 issue of CELL, we show that activation of TLR3 by retroviral vectors encoding the Yamanaka factors is critical for effective nuclear reprogramming of the human somatic cell to pluripotency (induced pluripotent stem cell, iPSC; see figure from Lee et al, Cell 2012).  TLR3 activation (as with viral constructs) leads to a cascade of events, including global changes in epigenetic modifiers, that places the chromatin into an “open configuration” (in a phenomenon we have termed “transflammation”). Now cognizant of this heretofore unrecognized phenomenon, we have combined a small molecule agonist of TLR3 with the Yamanaka factors as cell permeant peptides, to efficiently generate human iPSCs.   This approach avoids integrating viral vectors, and provides a method to generate therapeutic grade stem cells.  

We are now delving further into the mechanisms of this phenomenon, to determine if the global changes in epigenetic modifiers can be decoupled from the cascade of events triggered by TLR activation, i.e., we wish to identify a more specific activator of the global epigenetic changes required for the “open chromatin” configuration.  Such an activator would selectively place the human cell in a more plastic state, so that its phenotype might be therapeutically modified. 

Therapeutic transdifferentiation

The knowledge gained above is now being employed by our group to develop methods for “direct reprogramming”, i.e., for inducing transdifferentiation of one somatic cell to another.  Specifically, we have successfully transdifferentiated human fibroblasts into endothelial cells using a small molecule approach.  We intend to develop this methodology for transdifferentiating human fibroblasts to endothelial cells in vivo.  This methodology would have therapeutic applications in any form of healing.  For example, as fibroblasts migrate into the area of a myocardial infarction, they could be transformed into endothelial cells, which would form a microvasculature to provide the nutrition and niche for cardiac stem cells.  The intent would be to help patients heal with tissue, rather than with scar.

The effect of these small molecules could be facilitated by a nanoparticle strategy that will 1) enhance retention of the small molecules in the targeted tissue, so as to increase the local effect and to reduce off-target adverse events; 2) improve the pharmacokinetics and pharmacodynamics, e.g., with mechanisms for prolonged elution of the therapeutic agents.

Therapeutic areas-Pre-clinical phase

iPSC-ECs for peripheral vascular diseases

We have generated human iPSCs as described above, and have developed methods for differentiating them into endothelial cells (iPSC-ECs).  We have validated the endothelial nature of the iPSC-ECs by IHC, rtPCR and functional studies (figure).  Furthermore, we have shown that the human iPSC-ECs can generate capillaries in immunodeficient mice, and can improve perfusion in our murine model of peripheral arterial disease.  

We have studied the iPSC-ECs on nanopatterned collagen conduits developed by our collaborators at Stanford and Fibralign.  The iPSC-ECs align with the collagen fibrils, and these aligned cells are less adhesive for inflammatory cells than non-aligned cells.  

  The nanopatterned conduits are now being studied in models of lymphedema.  We have generated human lymphatic iPSC-ECs and have placed these on nanopatterned collagen threads to be implanted into a large animal model of lymphedema.  Fibralign is developing a catheter for implantation of the threads with our assistance, funded by a DOD grant.

Therapeutic areas-Pre-clinical phase

Extending the telomere for EC rejuvenation

Another approach to cardiovascular regeneration is to extend the telomeric DNA, so as to increase the replicative capacity and function of the cell.   We have developed a modified mRNA approach to transiently express telomerase in human somatic and adult stem cells.  Our approach increases telomere length and enhances replicative capacity of the cells.  Our novel RNA constructs are generated by the RNACore team directed by Dr. Cooke and managed by Dr. Roman Sukhovershin.  Our RNAcore is a core for the National Heart, Lung and Blood Institute (NHLBI) Progenitor Consortium, as well as the Cancer Prevention and Research Institute of Texas (CPRIT).  We provide RNA constructs of the Yamanaka factors, lineage determination factors, reporter constructs and genome editing tools, as well as customized constructs required by Consortium and CPRIT investigators, as well as scientists around the world.

We have proposed pre-clinical studies toward developing a new therapy for children with Hutchinson-Gilford Progeria Syndrome (HGPS). Cardiovascular disease is the major cause of illness and death in HGPS. The accelerated atherosclerosis that occurs in these children is likely due to vascular senescence. Senescent vascular cells are “atheroprone” in that they express adhesion molecules and chemokines that facilitate atherosclerosis, and generate increased amounts of reactive oxygen species that cause widespread aberrations in vascular function. We have evidence that that transient expression of telomerase can restore the replicative capacity and homeostatic functions of senescent endothelial cells from adult humans. We now propose to use mmRNA encoding telomerase to extend telomeres in senescent endothelial cells from children with HGPS. Our preliminary studies indicate that this therapy will restore the regenerative and homeostatic functions of the endothelium, thereby preventing or mitigating cardiovascular disease. Our group is performing the studies to assess proof-of-concept with hTERT mmRNA in cells derived from Progeria patients and we are collaborating with HMRI nanomedicine faculty to encapsulate mmRNA in nanoparticles for delivery.

Endothelial regeneration and nitric oxide (NO)

Healthy endothelial cells have a tightly regulated NO synthase (NOS) complex that generates NO in response to hemodynamic and humoral activation.  NO enhances endothelial survival and replicative capacity, and is a critical regulator of vascular homeostasis. In patients with cardiovascular disease or risk factors, NO synthesis/bioavailability is reduced, in part due to production of the endogenous NOS antagonist ADMA (asymmetric dimethylarginine).  ADMA is normally metabolized by the enzyme DDAH (dimethylarginine dimethylaminohydrolase).  The activity of DDAH is reduced in patients with cardiovascular disease.  We have developed novel activators of DDAH that should be useful in the treatment of insulin resistance and hypertension.  

However, there are conditions where overproduction of NO is pathobiological (such as sepsis and pulmonary fibrosis).  In these conditions, a DDAH antagonist would be useful.  In a high throughputscreen, we have found a number of excellent antagonists that have been validated by orthogonal assays

Therapeutic areas-Clinical phase

CD34+ BMMNCs for PAD

We are participating in the CCTRN study, which includes 7 national sites, funded by the NHLBI, to pursue clinical trials in adult stem cell therapy.  Our first trial will be using bone marrow derived CD34+ mononuclear cells (versus placebo) for IM injection in patients with intermittent claudication. The response to cell therapy will be extensively assessed, using treadmill exercise testing, MR perfusion imaging and MR angiography.  We hope to determine if ALDHbright (these are largely CD34+) BMMNCs enhance function and/or perfusion; in addition the cells will be extensively characterized using mass cytometry to look for biomarkers of success or failure.  
Adult stem cells for intermittent claudication and critical limb ischemia

We will participate in industry-sponsored studies of adult stem cells for IC and CLI.  The clinical research arm of the CCR will aggressively pursue opportunities to work with industry to assist in the execution of other adult stem cell trials for peripheral or coronary artery diseases.  Invaluable experience will be gained by this group in clinical trials, which can be leveraged later as the CCR develops its own regenerative therapies to the clinical stage.  Such studies will keep Methodist at the forefront of cardiovascular regeneration, and will provide our patients with cutting edge therapies.
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