Commentary on Some Recent Theses Relevant to Combating Aging: December 2020

Xiaolin Hu, PhD, Stanford University

Cells are the smallest functional unit of the human body, which contains 37 trillion cells on average. Current technology for intracellular sensing does not yet allow us to follow biological processes inside living intact cells on a continuous, real-time basis. Information about such processes is invaluable for understanding complex diseases, such as cancer, immune-pathological, and cardiovascular disorders. For example, the acidity in the micro-environment inside cancer cells can undergo transitory stages that last up to a few hours and are difficult to detect by non-continuous detection methods. However, most of today's diagnostic tests and medical monitoring operate at the body, organ, or tissue level. Continuous detection of cellular activities can significantly advance our knowledge of biology and disease and enable new discoveries in early diagnostics and tailored therapeutics.

This dissertation presents the exploratory work on a new approach toward intracellular sensing with chip-in-cell (CHIC) devices. Each CHIC device has a wireless front-end to communicate data and a sensor interface responsive to a specific stimulus. We present the design, fabrication, and characterization of the wireless front-end with a radio-frequency identification (RFID) and a corresponding external detector. The RFID diameter is 22 μm and can be naturally internalized by certain classes of living cells. This is the world's first cellular-implantable RFID. The detector can identify RFID presence and distinguish RFID capacitance changes via resonance frequency shifts. Requirements on system-level integration with biological microfluidic platforms are included.

To detect biologically meaningful parameters, a pH-responsive hydrogel is integrated as an example prototype CHIC sensor interface. The hydrogel is fabricated onto interdigitated electrodes. Capacitance shifts at gigahertz range are characterized in physiologically relevant pH ranges from 6 to 9, with steps as small as 0.25 pH identified. Overall, the CHIC sensors are addressable to individual cells, bio-compatible for long-term observation, and sensitive to specific chemical changes; a solid step toward continuous real-time intra-cellular sensing.

Comment: Recent decades have seen a surge of interest in single-cell analysis, thanks to an increasing appreciation of the heterogeneity of phenotypes present even in highly purified cell populations; when viewed in aggregate, this variance inevitably obscures the underlying biological processes in which those cells are engaged. Existing techniques to collect data from individual living cells with comparatively high throughput, such as fluorescence microscopy, are generally not well suited to continuous operation over extended periods of time—a capability that holds tremendous potential to uncover the causal and temporal relationships between intracellular phenomena. This dissertation introduces a genuinely remarkable proof of concept for sustained real-time intracellular monitoring, implementing on the micron scale a passive radiofrequency transponder encapsulated in biocompatible silicon dioxide and integrated with a pH sensor. (Although the physiologically relevant range for cytosolic pH is generally smaller than is given above, the achieved step size of 0.25 pH units is already sufficient to distinguish between normal cells—pH 6.9–7.1—and some cancer cells—which can reach pH 7.65.) Despite the fact that the 22 μm ring presented is relatively large compared with typical mammalian cells, it proves to be surprisingly well tolerated in initial tests with persistence (and viability) maintained for several days from inclusion into cells that are themselves of only marginally greater diameter! Smaller devices—down to 10 μm—are also considered, with notably better cellular internalization properties, but are found to be slightly beyond the limit of reliable interrogation with available detector technology. Indeed, a major limitation of this prototype is the very short range permitted between transponder and detector—on the order of a few micrometers—which requires microfluidic handling of the implanted cells to achieve the necessary positional accuracy. In addition, the equilibration time of the described sensor after a pH change is of the order of minutes, which is somewhat restrictive for the monitoring of fast cellular processes. None of these present shortcomings, however, dampens our enthusiasm for the fundamental advance reported here; given the typical pace of development in microelectronics, and the clear path for future development outlined in the conclusion of the study, we have no doubt that a host of devices with progressively superior performance will follow this exemplar in the near future.

John Lockhart, PhD, University of South Florida

The field of synthetic mRNA therapeutics is a rapidly expanding arm of gene therapies. The use of mRNA provides multiple benefits over viral or DNA vectors. Synthetic mRNA vectors are immediately translated into protein after entering the cytoplasm of cells in contrast to DNA vectors that must first be transcribed to mRNA in the nucleus. This allows synthetic mRNA to produce a therapeutic protein in any cell type, including non-dividing cells. In addition, the non-replicative nature of mRNA means that insertional mutagenesis or generation of escape mutants is not a concern. However, the stimulation of innate immune responses by unmodified synthetic mRNA prevented widespread clinical applications.

The discovery that incorporation of modified nucleotides, such as pseudouridine or 5-methylcytosine, prevents the recognition by innate immune sensors has renewed interest in the use of synthetic mRNA as a therapeutic. In conjunction, numerous post-transcriptional regulatory elements have recently been described in mRNA. Adding these regulatory elements to synthetic mRNA allows control of the expression of the encoded protein in tissue-, cell-, or environmental-specific conditions. However, the influence that the modified nucleotides commonly incorporated in synthetic mRNA have on the regulatory capacity of these elements has not been examined.

In this study we investigated what effects modified nucleotides have on the regulation of synthetic mRNA by microRNA (miRNA switch). We found that nucleotide modifications that increase the translation of the synthetic mRNA tended to decrease the regulatory capacity of microRNA switch. Inclusion of multiple microRNA target sites at the 3′ UTR of the synthetic mRNA was able to minimize the loss of miRNA-dependent regulation of the miRNA switch, but microRNA target sites complementary to the six-nucleotide microRNA “seed” sequence were more affected to nucleotide modification. We found the effect of nucleotide modifications varied between microRNA species and was not determined by the proportion of modified nucleotides present in the microRNA target sites. Finally, we observed that utilizing a single microRNA target site at the 5′ UTR of the synthetic mRNA completely ameliorated the loss of regulation due to nucleotide modifications.

Because synthetic mRNAs are easy to produce and can be made to encode any protein of interest, they are ideal for clinical development. Currently there are over 45 clinical trials underway utilizing synthetic mRNA as a monotherapy or in conjunction with other therapeutics. Most of these clinical trials are focused on cancer immunotherapy, particularly autologous T-cell therapy. This therapeutic modality is well suited for synthetic mRNA as the target cells are transfected ex vivo. This avoids the major obstacles that synthetic mRNA therapeutics must still overcome: delivery to target organs.

The sensitivity of synthetic mRNA to extracellular ribonucleases requires encapsulation of the mRNA in a protective nanoparticle. Numerous such nanoparticles have been reported, but nearly all are variations on either lipid nanoparticles or polymeric nanoparticles. The advances made thus far with these two mRNA delivery platforms have significantly reduced their toxicity, however the endosomal escape rate of these particles remains well below 5%. Furthermore, when administered systemically these nanoparticles are avidly taken up by sentinel macrophages of the liver and spleen or hepatocytes. The accumulation of particles in the liver has thus far limited the applications of mRNA therapeutics to diseases and disorders that are liver-specific or that can be treated by using the liver as a biosynthetic depot. Expanding the clinical application of synthetic mRNA may require the discovery of novel delivery platforms that are capable of targeting other organs.

In this study we also tested the delivery of synthetic mRNA using a small cell penetrating peptide, called p5RHH, that is derived from bee venom protein melittin. We showed that in the presence of mRNA, p5RHH self-assembles into spherical nanoparticles that display a high degree of RNase resistance. These nanoparticles were consistently sized regardless of the length of the mRNA payload. Furthermore, after uptake by cells, p5RHH-mRNA nanoparticles displayed a high degree of endosomal escape that was dependent upon the acidification of endosomes, which disassembles the nanoparticles. The high concentration of p5RHH in the lumen of the endosome led to efficient endosomal disruption and produces minimal cytotoxic effects. When the p5RHH-mRNA nanoparticles were injected intravenously into an atherosclerotic mouse, we observed robust expression of the payload mRNA in only the atherosclerotic plaques. The lack of expression in typical depot organs, such as the liver, spleen, lungs, or kidneys, was also confirmed in a normal mouse. The simplicity and specificity of p5RHH-mRNA nanoparticles makes them an ideal candidate for further pre-clinical development as an mRNA delivery platform.

Comment: Nearly 20 years passed between the characterization of messenger RNA by Sydney Brenner's group in the early 1960s and the first biomedical application of such molecules, in 1978, when an antisense transcript to the Rous sarcoma virus was employed to block its replication in cultured cells and thereby prevent their oncogenic transformation. Major milestones followed in the 1990s, including the demonstration that injected mRNA was in and of itself sufficient to drive protein expression in mouse skeletal muscle, and that pathogenic proteins could be similarly expressed to trigger an immune response (laying the groundwork for the development of RNA vaccines), the discovery of RNA interference, and the first regulatory approval of an RNA-based drug (fomivirsen, an antisense oligonucleotide that suppresses cytomegalovirus transcripts to prevent complications of the normally benign infection that otherwise manifest in immunocompromised patients). After that pulse of activity, the field experienced something of a lull until the second decade of the new millennium, with only one further drug approved in 15 years (although that one drug—pegaptanib—did illustrate a novel mode of action, using an aptamer structure to bind to and block the function of an endogenous protein). Despite the translational hiatus, a key discovery was made during this period—that the inclusion of certain chemically modified nucleotides in RNA transcripts can mask their presence from the innate immune system, neutralizing the autoimmune pathology observed in many earlier studies. Thanks, in part, to that development, seven further drugs have been approved in the past 7 years, with dozens more now undergoing clinical trials. This dissertation identifies a general negative consequence of such modifications to the transcript structure—an accompanying loss of regulatory control by microRNA, which can compromise the specificity and potency of expression—but, astonishingly, finds that this consequence can be rescued simply by switching one miRNA target site from its typical 3′ UTR location to the 5′ UTR. The author then addresses the largest single barrier to wider application of RNA therapeutics: targeting to sites other than the professional phagocytes that avidly and nonspecifically take up nanoparticles from the bloodstream. Only two other populations have been effectively targeted to date in vivo (hepatocytes through the asialoglycoprotein receptor, and pancreatic β cells through the glucagon-like peptide-1 receptor); the discovery herein of a nanoparticle class that robustly eludes the body's professional phagocytes and instead accumulates preferentially in areas of the circulation where the endothelial barrier is disrupted will be of tremendous value in the deployment of RNA therapy against highly prevalent cardiovascular diseases.

Yujia Yue, PhD, Temple University

Cardiovascular disease remains the leading cause of morbidity and mortality worldwide and Myocardial Infarction (MI) and subsequent heart failure remains the leading cause for death. Despite the improvement in prognosis and treatment of acute MI patients, the underlying causes including loss of cardiomyocytes and microvasculature remain potential risk and lack proper and efficient solutions. Stem cell-based therapies for repair and regeneration have evolved and have been applied in clinical trials. Different types of stem cells, including Endothelial progenitor cell (EPC), Mesenchymal Stem Cell (MSC), induced Pluripotent Stem Cell (iPSC) and cardiac progenitor cells etc. have been used for potential long term recovery and cardiac regeneration. However, results from the clinical trials have been largely disappointing and improvement in cardiac functions have been modest likely due to the limitations of cell therapy including low integration in myocardium, poor survival, cellular dysfunction and limited differentiation ability. It is therefore necessary and urgent to develop cell free alternatives as next generation regenerative therapies. There is a consensus that the beneficial effect of stem cell therapy is largely due to paracrine effects. Exosomes have recently emerged as important functional units mediating stem cell paracrine effects. Exosomes are the family of extracellular vesicles (EV) which are 30–150 nm in size, secreted by almost all types of cells and responsible for cell-cell communication via delivering their cargo including RNAs and proteins to host cells. Studies from our and other labs have shown that exosomes mimic parental stem cell in improving post-MI functions. The essential feature of exosome is decided by their cargo including RNA and protein, which are subject to dynamic changes depending on the environment of parental cells. Our studies were focused on Endothelial Progenitor Cell (EPC)-derived exosomes. EPCs are generated in bone marrow, and home to the site of tissue injury and orchestrate neovascularization and tissue repair. Patients with ischemic heart disease, are usually accompanied with comorbidities such as systemic inflammation, aging, diabetes, etc. which are known to compromise EPC functions. We hypothesized that EPCs under inflammatory stress produce dysfunctional exosomes with altered RNA and protein content, leading to impaired cardiac reparative properties. We chose interleukin-10 knockout (IL-10KO) mice as a model of systemic inflammation. EPCs were isolated from IL-10KO and wild-type (WT) mice, and their exosomes (Exo) were compared for their reparative properties both in vitro and in vivo. Our in vitro studies showed WT-EPC-Exo treatment attenuated recipient cell apoptosis, enhanced cell mobilization and tube formation, whereas IL-10KO-EPC-Exo were functionally deficient or even had detrimental effects. We used MI mouse model to compare the in vivo function of two groups of exosomes and found WT-EPC-Exo treatment significantly improved left ventricular (LV) cardiac function, inhibited cell death, promoted angiogenesis and attenuated cardiac remodeling; while these cardioprotective effects were lost in IL-10KO-EPC-Exo treated group. Both in vitro and in vivo studies proved that even the same progenitor cell type (EPCs), under inflammatory stimulus (IL-10KO), secretes exosomes with different reparative properties. Next, we explored whether the observed difference in exosome function is caused by altered exosome content. Using Next Generation RNA Sequencing (NGS RNAseq) and mass spectrometry we found RNA and protein expression patterns were drastically different in wild type and IL-10 knockout EPC derived exosomes. This evidence leads to the conclusion that alteration in exosome content is fundamental for exosome function. We picked two candidates that are highly enriched in IL-10KO-EPC-Exo for further study, miR-375 and Integrin-Linked Kinase (ILK). We treated IL-10KO-EPC with anti-miR against miR-375 and siRNA against ILK separately, and successfully decreased the expression of miR-375 and ILK in both EPCs and EPC derived exosomes. Then we explored the function of those miR and protein “modified exosomes” with similar in vitro and in vivo experiments as previously described. Compared to IL-10KO-EPC-Exo, miR-375 knockdown exosomes showed enhanced angiogenesis and inhibited cell apoptosis, while ILK knockdown in exosomes rescued functions in both in vitro and in vivo experiments. These results suggested the possibility that exosome manipulation of identified factors may partially rescue their reparative functionality.

In summary, our studies revealed that stem cell derived exosomes are capable for independent cardiac repair in ischemic heart disease, however, parental stem cells under pathological stimulus secrete dysfunctional exosomes with altered RNA and protein content. Exosome function can be rescued or enhanced through RNA and protein content modification.

Comment: Since the characterization in the 1960s of hematopoietic stem cells and the discovery of their remarkable capacity to repopulate the immune system, bone marrow transplants have become a routine clinical practice and have drastically improved the prognosis of patients taking chemotherapeutic drugs. However, although many other lineages of stem cells have now been identified, their therapeutic application has largely foundered (notwithstanding claims to the contrary by a frankly worrying number of opportunists). In particular, the fundamental assumption that such cells when transplanted would differentiate and replace missing somatic cells in vivo, leading to a permanent cure, has proven unreliable—with a majority of the benefits observed in preclinical studies now attributed instead to factors secreted by the stem cell population, rather than the cells themselves. Curiously, that phenomenon is notable even in cases where the patient's body naturally contains a significant population of the same progenitor cells; thus we might ask why newly transplanted cells are able to drive repair when resident cells of the same class are not. Recent study has indicated that although freely diffusing mediators are involved in the regenerative secretome, a more significant role may be played by exosomes—membrane-bound vesicles that selectively incorporate a range of proteins and nucleic acids, and that are increasingly recognized as crucial in intercellular communication and regulation. Importantly, exosomal content in general is known to vary substantially according to the current metabolic state of the originating cell, as well as its developmental lineage. This dissertation explores the role of exosomes in the repair of cardiac damage by progenitor cells, an application that has shown particularly disappointing results in clinical trials, finding a marked deficiency in the regenerative potency of such particles when derived from cells subjected to sustained inflammatory stress. Since chronic inflammation is a hallmark of the aged or diseased body, these results may explain how it is that freshly transplanted progenitor cells can orchestrate healing far more effectively than their resident equivalents—yet do so only transiently (until they too fall victim to the inflammatory environment)—as well as the anomalous success of hematopoietic stem cells (which are of course best placed to persistently improve that environment). Engineering transplant-ready cells to resist such interference—or indeed finding a method to install the same resilience in resident progenitors—may allow us to realize much more of the original promise of stem cell therapy.

David Lam, MS, University of California, San Diego

There is an exponential increase in the incidence of disease with age such as cancer, cardiovascular disease, and neurological disorders. Investigating novel approaches to address aging should be given serious consideration since age is a major common risk factor for chronic diseases. Aging and development of progeny to adults has typically been considered a unidirectional process. However, that notion is no longer clear since terminally differentiated cells can now be reprogrammed to an embryonic-like cell—termed induced pluripotent stem cells (iPSC). This suggests that at the cellular level, development is not limited to one direction and can be reversible. Lapasset and colleagues conducted experiments showing improvements in markers of aging when fibroblasts from centenarians were reprogrammed to iPSCs and subsequently re-differentiated into “rejuvenated” fibroblasts. This experiment and others suggested that reprogramming rejuvenated the cells. However, it remains unclear when the “rejuvenation” occurred—during the reprogramming process or when the cell was pluripotent. Here, we show that partial reprogramming rejuvenated cells through several reduced hallmarks of aging. Furthermore, partial reprogramming via short term cyclical induction of Yamanaka factors also extended the lifespan of our animal model. Reprogramming may be useful for understanding the molecular basis of aging and this study is a proof of concept that reprogramming has the potential for therapeutic application.

Comment: Although the human genome is in general remarkably well protected from mutation over the lifespan, it is now well established that the same cannot be said for the epigenome—the collection of chemical modifications that decorate both DNA and the histone proteins that package it within the nucleus. Epigenetic marks can profoundly alter gene transcription, and directed modification of the epigenome is a fundamental mechanism by which cells regulate their identity and metabolic state. As cells age, the lability of the epigenome permits a gradual accrual of stochastic alterations, the collective impact of which tends to impair cellular function. (The term “epimutation” is sometimes used to describe all such changes, but properly refers only to the small subset of them that are heritable.) Indeed, the relationship between this accumulation and health is so strong that epigenetic “clocks”—algorithms that estimate agedness based on a subset of such factors—can in some cases predict the risk of age-related disease and mortality more accurately than chronological age. Of course, children do not begin life at the biological age of their parents: it was always clear that the process of embryonic development would include some means to reset the epigenetic state of the cell. Nonetheless, achieving the same clean-up in an existing organism was a herculean prospect even in theory, thanks particularly to the absence of any reliable reference copy of the code—there is no analogue at the epigenetic level to the parity check provided by nucleotide base pairing, and the “correct” sequence is heavily dependent on cell type and circumstances. Shinya Yamanaka's Nobel-winning discovery of induced pluripotency in 2006 led to the surprising realization that an epigenetic reset could occur in single adult cells, entirely decoupled from the complex processes of gametogenesis, and thus for the first time raised the possibility that age-related epigenetic decline might realistically be susceptible to redress. Unfortunately, although the induction of pluripotency followed by redifferentiation to the prior lineage can indeed profoundly rejuvenate cells from even the very elderly, initial attempts to induce pluripotency in vivo have displayed a worrying tendency to also induce teratomas. Recently, encouraging evidence has been presented that the loss of somatic gene expression (dedifferentiation) that occurs during pluripotency induction follows distinct kinetics to the epigenetic remodeling triggered by the same stimulus, implying the potential for the two to be disentangled. This thesis reports on a body of work conducted at the Salk Institute over the past few years, showing that transient expression of the Yamanaka factors can indeed decouple the two phenomena and achieve a “partial reprogramming” in which cellular identity is not lost and there is little or no measurable increase in the risk of malignant transformation, but measures of epigenetic age still decrease. (One important caveat is that although apparently insufficient to drive carcinogenesis de novo, partial reprogramming can promote the development of invasiveness in pre-existing early stage cancers.) Importantly, this epigenetic rejuvenation is not highly persistent—long-term benefits are observed only with cyclical application of the reprogramming stimulus. Although therapeutically problematic, this is actually something of a relief from a strategic perspective: it implies that changes to the epigenome play an intermediary role in age-related pathology rather than being a root cause. Whether or not partial reprogramming can be refined to achieve a safety profile acceptable to clinicians remains to be seen but, even if not, there is cause for continued optimism that resolution of the molecular damage that provokes epigenetic disorder could ultimately eliminate the need to tackle it head-on.

Erika Williams, PhD, The University of Mississippi Medical Center

The aim of this project was to evaluate the renal structural and functional outcomes following a novel therapeutic strategy in renovascular disease (RVD), a progressive disease that causes deterioration of renal function, increases cardiovascular risk, and may progress towards the development of chronic kidney disease (CKD). Previously published pre-clinical and clinical data indicate that mechanical resolution of the vascular obstruction that initiates the disease via renal angioplasty (PTRA) is often not successful at improving renal outcomes and resolving hypertension in RVD. These observations highlight a dire need for new treatment strategies.

Our previous work using a translational swine model of unilateral RVD showed that the progressive deterioration of stenotic kidney function and development of renal fibrosis associated with progressive microvascular (MV) rarefaction in both the cortex and the medulla. Interestingly, these pathological events associate with a progressive decrease in the renal expression and availability of vascular endothelial growth factor (VEGF), an angiogenic cytokine with pivotal roles in the maintenance and repair of MV networks everywhere, including the kidney. Evidence from our laboratory also showed that replenishment of VEGF to the RVD kidney is able to significantly recover renal hemodynamics and MV integrity while reducing renal injury and hypertension. Furthermore, subsequent studies from our laboratory aimed to refine renal delivery of VEGF in RVD by using elastin-like polypeptides (ELPs) as a drug delivery vector. The ELP carrier was fused to VEGF to design a novel ELP-VEGF construct, which shows high affinity for the kidney and a longer circulating time than VEGF that is not fused to a carrier (“free” VEGF). We recently demonstrated that experimental ELP-VEGF therapy had superior effects on renal hemodynamics, MV structure and function, tissue injury, and hypertension than administration of free VEGF.

Taken together, these data suggest that the renal microvasculature beyond the initial vascular obstruction plays a pivotal role in defining the progression and recovery of renal function and injury. Therefore, the overall hypothesis of this project was that using a co-adjuvant approach to treat RVD by combining PTRA with ELP-VEGF administration would produce better renal outcomes than PTRA alone and confirm the importance of the integrity of the renal microvasculature in RVD progression. We further hypothesized that we could replicate our experimental findings by designing and applying a mathematical model of the known and hypothesized components of MV rarefaction in RVD to confirm their important pathophysiological roles and outcomes of renal MV protection after targeted therapies.

One of the major experiments was designed to test the therapeutic efficacy of combined PTRA and ELP-VEGF therapy in an experimental model of RVD. Using a clinically relevant swine model of RVD (achieved by unilateral renal artery stenosis and high cholesterol feeding), PTRA with stenting (PTRAs) was performed simultaneously with a single intra-renal injection of ELP-VEGF (or placebo) after six weeks of RVD and established renal injury and hypertension. Renal hemodynamics were assessed in vivo by multi-detector computed tomography (MDCT) immediately before treatment and again four weeks after treatment in order to assess the impacts of the therapeutic intervention. Following in vivo experiments, ex vivo micro-CT was used to evaluate the impact of the therapy on the structure of the renal microvasculature, and renal expression of proteins involved in MV proliferation, repair, and damage, and renal fibrosis were quantified. Our study showed that PTRAs+ELP-VEGF therapy improved stenotic kidney hemodynamics and function, reduced markers of renal injury, and improved renal angiogenic signaling, MV density and remodeling, and fibrosis more efficiently than PTRAs alone. These results indicated that the greater improvements in renal function when ELP-VEGF is administered with PTRAs compared to PTRAs alone may be driven by improved MV proliferation and repair, which ameliorates MV rarefaction and fibrosis distal to the stenosis that cannot be improved by PTRAs alone. This study supports a potential novel treatment strategy for RVD and the notion that renal injury may eventually become irreversible and non-responsive to PTRAs alone. Furthermore, this study highlighted the importance of VEGF for renal MV integrity and the role that renal MV protection plays in the recovery of the RVD kidney.

The other major experiment was designed to determine whether a mathematical model of the known and hypothesized pathophysiology of renal MV deterioration in RVD can predict the experimental outcomes we observed in the swine RVD model and confirm the importance of the renal microvasculature in disease progression and resolution. For this complementary study, we created a Boolean model of factors known to contribute to the progression of RVD. A Boolean model is a type of discrete model that describes qualitative aspects of a biological system. At any given point in time, each component of the system can be either fully on/active or off/inactive. We created a network of 20 factors known to be involved in the progression of RVD that allowed simulation of 5 therapeutic interventions that were previously tested in the swine RVD model, including co-adjuvant PTRAs+ELP-VEGF. Each factor was assigned a function based on its interactions with other components of the model, and simulations of interventions were performed until outcomes reached a steady state and then analyzed to determine the pathological processes that were activated, inactivated, or unchanged compared to RVD with no intervention performed. The Boolean simulations matched the results of our previous experimental studies, confirming the importance of the integrity of the renal microvasculature on treatment outcomes in RVD. Importantly, the Boolean model predicted greater improvements in renal outcomes with combined PTRAs+ELP-VEGF than with PTRAs alone, as we observed in the swine model of RVD. This study helped to solidify our current understanding of the pathophysiology of renal MV deterioration and RVD progression and further supported the efficacy of PTRAs+ELP-VEGF therapy to restore renal function and repair.

Taken together, these two experiments extend our previous findings on the role of the renal microvasculature in the pathophysiology of RVD and support the notion that co-adjuvant PTRAs with ELP-VEGF therapy is a feasible and efficacious approach to ameliorate renal injury in RVD using a multi-targeted approach, which may open new avenues for future studies to extend the application of this strategy to other forms of renal disease in which renal MV deterioration is involved.

Comment: The mammalian circulatory system is the principal route by which cells exchange the molecular feedstocks and waste products of metabolism, as well as the chemical signals that permit coordinated multicellular life. Any impediment to that flow of material places a host of stresses on the affected tissues; this is most clearly observed in cases of complete vascular obstruction, where urgent medical care is needed to avoid rapid hypoxic cell death (not to mention, in many cases, systemic death). A much less drastic but relentless circulatory stress occurs throughout aging bodies, thanks to a general decline in the biophysical function of the vasculature, the culmination of factors including chronic inflammatory signaling, ectopic deposition of fats and minerals, and unresolved damage to the long-lived proteins that make up the extracellular matrix. This diffuse pathology is now recognized to play an important role in a broad spectrum of age-related diseases, with negative impacts eventually observed in virtually every organ system. Although most obstructions to the major vessels can potentially by repaired by surgical intervention, it is rare for such treatments—even when maximally successful—to lead to a full recovery of function, except perhaps in extremely young children. This dissertation adds to a growing body of work demonstrating that this failure is attributable at least, in part, to residual microvascular damage downstream (hemodynamically and causally) of the primary injury, and further shows that simply introducing an appropriate growth factor can significantly reverse that secondary deterioration and restore tissue function. While grounds for cautious optimism, it must be noted that the model employed does not recapitulate the broader regenerative impairments of the aged milieu; further work is required to establish whether VEGF signaling alone can drive clinically meaningful renal recovery in older subjects, although the prevalence of chronic kidney disease (which causes over a million deaths annually, and drastically increases health care costs for around three-quarters of a billion people worldwide) surely justifies a prompt start to that investigation. In the longer term, growth factor supplementation—with its almost unavoidable potential to accelerate the development of cancer—is a somewhat risky practice. Ideally, the situation should be remedied in the first instance by comprehensively repairing the molecular damage that underlies vascular dysfunction, which will at the very least minimize the magnitude of the proliferative stimulus required to fully restore the microvascular network (if not obviate it entirely). Therapies to tackle the accumulation of senescent cells—one facet of that damage—are under very active clinical investigation at present, notably including at least one trial specifically addressing diabetic kidney disease; the data from that investigation should help clarify the extent to which renal microvascular deterioration is capable of self-correction given favorable conditions to do so.

Saketh Karamched, PhD, Clemson University

Cardiovascular diseases (CVDs) are the leading cause of death globally. An estimated 17.9 million people died from CVDs in 2016, with ∼840,000 of them in the United States alone. Traditional risk factors, such as smoking, hypertension, and diabetes, are well discussed. In recent years, chronic kidney disease (CKD) has emerged as a risk factor of equal importance. Patients with mild-to-moderate CKD are much more likely to develop and die from CVDs than progress to end-stage renal failure. Vascular calcification (VC), typical in aging, several genetic and metabolic disorders, is now recognized as a strong and independent predictor of cardiovascular events and mortality, not only in diabetic and CKD patients, even in the general population. VC is classified into two distinct types based on location in the vessel wall; intimal and medial. Elastin-associated medial arterial calcification (CKD) is more specific to CKD and contributes significantly to cardiovascular mortality in these patients. It is responsible for loss of vessel elasticity, increased arterial stiffness, increased pulse pressures and systolic blood pressure, and left ventricular hypertrophy ultimately causing arrhythmias and heart failure.

Current clinical practice is mostly focused on prevention and retardation of VC progression. Unfortunately, most patients with CKD remain underdiagnosed, and those diagnosed have already heavily calcified vessels. As such, they are undertreated since preventative strategies no longer work at this stage. Unfortunately, there is no FDA-approved treatment available that reverses calcification in countless CKD patients. A treatment strategy which promotes resorption of calcified lesions, while simultaneously avoiding demineralization from normally calcified tissues (i.e., bones and teeth) remains an urgent health care need. Chelating agents bind to metal cations, can dissolve and “wash away” calcium deposits if delivered in close proximity to the calcification sites.

This work was undertaken to see if we can develop targeted therapies to deliver chelating agents to vascular calcification sites. Amongst chelating agents known for their affinity to Calcium ions (Ca²⁺), we found that EDTA chelates Ca²⁺ from hydroxyapatite better than others. In our laboratory, we have developed a unique targeting mechanism by using nanoparticles to deliver chelating agents and other drugs to degraded elastin, a characteristic feature of VC. We take this approach forward in clinically relevant animal models of CKD.

First, we tested the targeted nanoparticle-based EDTA chelation therapy in a rat model of adenine-induced renal failure. The targeted nanoparticles delivered EDTA at the sites of vascular calcification and reversed mineral deposition without any side effects. Furthermore, we validated the adenine-CKD model in mice to monitor MAC in vivo and explore the phenotypic and functional alterations associated with it. We were able to target our nanoparticles to calcified arteries in these mice. The mouse model will help us to test whether our EDTA chelation therapy tangibly improves arterial function by restoring vascular health. Lastly, we investigated the possibility of using an ex vivo organ culture model of VC as a simpler, and relatively easier model to assess if EDTA chelation therapy promotes vessel homeostasis. The work presented here represents another major step forward towards the development of targeted EDTA chelation therapy as an unconventional therapeutic approach to reverse pathological calcifications in CKD patients.

Comment: The association between cardiovascular disease and calcification of the circulatory system was first described in the 19th century, and the extent of calcification is now recognized as a quantitative predictor of negative outcomes in several chronic diseases. This is to a large extent a straightforward biophysical consequence; the elasticity of the vasculature is critical to both its effective function and its resilience, and it is no surprise at all that the introduction of rigid crystalline deposits compromises that property. In the most prominent such condition—atherosclerosis—calcification is thought to be secondary to the presence of excess oxidized lipids in the vessel wall; there is some evidence that natural mineral balance can reassert itself once those toxic species are cleared, at least in relatively immature plaques (although the technology to achieve this clearance remains under development). Conversely, the calcification observed in kidney disease appears to result directly from the derangement of serum metabolites that occurs in the absence of a fully functional filtration system, and there is little evidence of any inherent capacity for resolution while that situation persists. Slow progress in the development of treatments to reverse kidney dysfunction has prompted considerable interest in the interim strategy of periodically removing the surplus calcium. The efficient extraction of calcium from biological substrates by chelation (high-affinity coordinate bonding of a ligand to a metal ion) is straightforward enough—indeed, it is ubiquitously employed in laboratory procedures to regulate the concentration of the element—but when such agents are applied systemically, the positive impact on the progression of vascular lesions is invariably outweighed by detrimental effects on the skeleton. Furthermore, there is still considerable uncertainty over how rapidly deposits cleared by chelation might reaccrue after therapy—and hence whether such an agent could ever be deployed on a dosing schedule compatible with wide clinical use. This thesis builds on a history of work in the host laboratory to demonstrate that targeting the workhorse Ca²⁺ chelator EDTA to areas rich in degraded elastin—specifically distinctive of vascular lesions—efficiently reverses established calcification in rodent models of chronic kidney disease with no detectable systemic side effects. Crucially, calcification was observed not to return even after a 4-week interruption of treatment—a finding that bodes well for the translation of this form of therapeutic to practical medical applications.