The Scientific Journey to Investigate and Validate the CVAC Hypotheses rev 2 0 1 08 22 2015

The Scientific Journey to Investigate and Validate theCVAC Hypotheses

- an abridged discussion

By Allen J. Ruszkowski

Once a discovery is made and a hypothesis is constructed generally the first avenue to be explored in the search for the truth to validate the hypothesis is to conduct a review of the published scientific literature for evidence to support the validation or invalidation of the hypothesis. Over the last 200 years our Civilization has made huge investments into research studies of the diseases associated with allostatisis, allostaticload and the balance between energy input and expenditure. These diseases include Diabetes, Alzheimer’s, Arthritis, Heart Disease and Cancer. We therefore expected that there should some evidencein the published scientific literature that could guide our way. This discussion is intended to highlight some of the critical keyscientific markers we discovered along this rocky road that we are traveling that have guided our pathways.

In 1987, shortly after Carl Linton observed the improvements in health status of individuals exposed to short stays at simulated altitude, he began to experiment with changing the rate at which the air was reintroduce into the pressure vessel. He observed a positive relationship with the rate of repressurization with the rate of improvement in health status. This provided a clue to the possibility that these benefits were the result of changes inpressure in addition to the low pressure environment and intensely piqued his interest to find out why. One of the technologies available to him was a device that could measure the

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 1

voltage on the surface of the cell membrane. So he constructed anexperiment where he subjected cells to low pressure and changes in low pressure and measured a resulting change in voltage. At critical pressure points, reversals in polarity were observed. This piezioelectric effect of the nature of the cell membranes has since been published by Jalki1 in 2007.

1 A. Jakli, J. Harden, C. Notz, C. Bailey. (2007). Piezoelectricity of phospholipids:A possible mechanism for mechano-, and magneto-receptions in biology. electronic-Liquid Crystal Communications April 10, 2007 : http://www.e-lc.org/docs/2007_04_09_12_01_56

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 2

This discovery then launched Carl Linton to conduct more than 10,000 hours of study to review the published scientific researchregarding short stays at simulated altitude. To many peoples’ surprise there exists publications of studies dating back to the 1930s regarding some of these effects. Carl also spoke with someof these researchers that had published these data. This included Dr. Z. I. Barbashova2 one of the first scientists to publish on this work.

Carl Linton, having been trained in one of the first education programs for the then new methods encompassing Respiratory Therapy, developed physiological constructs that would predict the appropriate application of pressure changes to induce beneficial physiological effects while avoiding detrimental effects. He developed a human and equine pressure vessels and experimented with these techniques on himself, eager athletes andrace horses. One method he utilized was the “Pinking Up” of humans and the mood improvements of the race horses to fine tune his algorithms for the application of the changes in pressure. These methods were pursued as a proof of concept to obtain funding for formal research along this pathway.

In 2003, as CVAC Systems, Inc. was being formed, Carl Linton was introduce to Allen Ruszkowski, a medical device entrepreneur of considerable accomplishments in relevant areas such as preventionof heart disease and hypertension. Allen Ruszkowski then began his own Journey to investigate the published scientific studies relevant to the CVAC Process. He spoke with Mike Nichols MD the CEO of the Tempus Clinic in Los Gatos, Donald Chu PhD the head ofPhysical Therapy and Athletic Training at Stanford University andCraig Cisar PhD a senior Kinesiologist at San Jose State

2 Z. I. Barbashova, G. I. Gregor’ yeva. (1969) Dynamics of change in the osmotic resistance of erythrocytes in humans and in animals during the period of natural acclimatization to high altitude (July 1, 1969). : https://archive.org/details/nasa_techdoc_19690022774

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 3

University. Based on those discussions he chose to join Carl Linton on this Journey. At that time adaptation to high altitudeand athletic performance / endurance enhancements were key strategic initiatives for the US Government DARPA research due tothe invasion of Iraq and Afghanistan. This provided an avenue forformal research funding and the first publication3 of the formal research study by researchers at the University of Hawaii in elite athletes demonstrating a very significant increase in the oxygen carrying capacity of the blood. This study also measured an increase in oxygen absorption (VO2 max) of more than 5% in responding elite athletes. This study along with the replicationof those data by researchers at Stanford University and others are the basis of the claims that the CVAC process improves fitness.

These data from the University of Hawaii, prior to publication, opened the door to other scientists, such as Anne Friedlander PhDat Stanford University. Concurrent with her research in the effects of the CVAC process in elite athletes, she created and taught a human biology course at Stanford University for Junior and Senior biology students focused on the CVAC Process and the specific resulting challenges faced by life science entrepreneurs.

The students were challenged to scour the published science relating to the CVAC Process and underlying mechanisms and in thefirst few weeks of the course take a position that the CVAC Process is either an effective new life science modality or an ineffective scam and then design a research study to prove their point for one of the potential disease or fitness applications. 3 Hetzler RK, Stickley CD, Kimura IF, et al. (2009). The effect of dynamic intermittent hypoxic conditioning on arterial oxygen saturation. Wilderness Environ Med 20:26–32. : http://www.ncbi.nlm.nih.gov/pubmed/19364183http://www.wemjournal.org/article/S1080-6032(09)70080-8/pdf

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 4

During the first few weeks of the course during this decision process the students complained that there was an inadequate amount of published data to make a decision. It was pointed out to them this is exactly the problem faced in the real world by life science entrepreneurs.

The course proceeded with students stating this was their most challenging academic experience. Preliminary positions were takenwith 50% / 50% decisions on each side of whether the CVAC Processwas an effective new modality or just another scam. Then within a few weeks a team of two students discovered and reported to theclass studies relating incident pressure cycling and the induction of the nitric oxide signaling mechanism. The teams in the class that had taken the position that the CVAC process was ascam, reversed their position to “the CVAC Process being an effective new modality”. The class was then unanimous in their positions and focused on designing research protocols to prove their points for different potential applications. In the post-course / instructor reviews many of the students commented that it was the best class that they had taken in the Human Biology Program at Stanford University. One student proclaimed that this class caused him to change his career from MD to PhD as a result.

This nitric oxide mechanism and its relationship to pressure cycling and mitochondrial biogenesis4 became a new avenue of investigation in the pursuit of our validation. Also at that timestudies began to be found linking altitude exposure and glucose

4 Nisoli E1, Carruba MO. (2006) Nitric oxide and mitochondrial biogenesis. J Cell Sci. 2006 Jul 15;119(Pt 14):2855-62. : http://www.ncbi.nlm.nih.gov/pubmed/16825426

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 5

metabolism in rats5 and in humans6. This development became a major signpost on our path indicating that the CVAC process may be improving insulin sensitivity and reducing its inverse: insulin resistance.

In 1988 Gerald Reaven delivered the Banting Lecture7,8 which

dramatically energized the Endocrinology Research Community. In his lecture, he revived a 150 year old theory that all age related diseases are caused by the same underlying mechanism and put forth the theory that the underlying mechanism was related toinsulin resistance. In 2001 researchers on his team published data from a prospective outcome study linking insulin resistance to T2 Diabetes, Hypertension, Heart Disease, Stroke and Cancer9:

5 Li-Ling Chiu, Shih-Wei Chou, Yu-Min Cho, Hsin-Yi Ho, John L. Ivy, Desmond Hunt, Paulus S. Wang, Chia-Hua Kuo. (2004) Effect of Prolonged Intermittent Hypoxia and Exercise Training on Glucose Tolerance and Muscle GLUT4 Protein Expression in Rats, J Biomed Sci 2004;11:838–846 : http://www.ncbi.nlm.nih.gov/pubmed/15591781

6 Lee WC, Chen JJ, Ho HY, Hou CW, Liang MP, Shen YW, Kuo CH. (2003) Short-termaltitude mountain living improves glycemic control. High Alt Med Biol. 2003 Spring;4(1):81-91.: http://www.ncbi.nlm.nih.gov/pubmed/12713715

7 Gerald M Reaven. (1988) Role of Insulin Resistance in Human Disease. Diabetes (December 1988) 37:12 1595-1607 : http://diabetes.diabetesjournals.org/content/37/12/1595.full.pdf

8 http://en.wikipedia.org/wiki/Banting_Lectures

9 Facchini FS1, Hua N, Abbasi F, Reaven GM. (2001) Insulin resistance as a predictor of age-related diseases. J Clin Endocrinol Metab. 2001 Aug;86(8):3574-8. : http://www.ncbi.nlm.nih.gov/pubmed/11502781

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 6

These data were particularly important to us because the number for age related disease in the lower third tertile was zero. As acomparison it seemed to us insulin resistance is far more predictive than LDL cholesterol (bad cholesterol) where 1/3 of people with high LDL never develop heart disease an 1/3 of peoplewith low LDL develop heart disease. Cholesterol lowering medications are a multi-billion dollar industry. There is also evidence that insulin resistance is modifiable. The Cardiology Community was promoting awareness and intervention labeling metabolic syndrome as a way to attack insulin resistance.

At this stage we then again sought out some of the most highly respected and open minded scientists, provided these data, and asked their advice. Both Mike Nichols MD at the Tempus Clinic andAnthony De Maria MD Chief of Cardiology at the University of California San Diego and editor of the Journal of American College of Cardiology advised us to investigate metabolic syndrome and insulinresistance. Dr. De Maria also suggested heart failure as a possible avenue for investigation because the data we showed him,including the CVAC specific data in elite athletes, demonstrated that the CVAC process was improving fitness. He indicated that it had been adequately proven that external counter pulsation (EECP) is effective in heart failure and that 100% of the benefitis obtained from improved fitness. However, the economic failure

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 7

and obstacles faced by the manufacturers of EECP would have made obtaining funding for research in this area highly unlikely from life science venture capitalists. So we chose to initiate the first blinded placebo controlled study of the CVAC Process in individuals at risk for metabolic syndrome10.

During the pursuit of this research for preventing metabolic syndrome we found work published from researchers at Yale University and Children’s Hospital in Boston linking insulin resistance with a 36% mitochondrial deficit11. With the press surrounding the initial public offering of Sirtris Pharmaceuticals which garnered a $320 million dollar valuation with data in mice and a $720 million dollar buyout by Glaxo SmithKline, insulin resistance and mitochondrial biogenesis were clearly an avenue worth investigating. The success of diabetes reversals with bariatric surgery also brought our attention to calorie restriction. Calorie restriction has been associated withlonger lifespan in yeast, worms and mice. We found a study where humans experimenting with calorie restriction had a 36% greater quantity of mitochondria12.

10 Marquez JL1, Rubinstein S, Fattor JA, Shah O, Hoffman AR, Friedlander AL. (2013) Cyclic hypobaric hypoxia improves markers of glucose metabolism in middle-aged men. High Alt Med Biol. 2013 Sep;14(3):263-72. doi: 10.1089/ham.2012.1057. Epub 2013 Sep 12. : http://www.ncbi.nlm.nih.gov/pubmed/24028640

11 Katsutaro Morino, Kitt Falk Petersen, Sylvie Dufour, Douglas Befroy, Jared Frattini,Nadine Shatzkes, Susanne Neschen, Morris F. White, Stefan Bilz, Saki Sono,Marc Pypaert, and Gerald I. Shulman. (2005) Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents. J Clin Invest. 2005 Dec;115(12):3587-93.Epub 2005 Nov 10. : http://www.ncbi.nlm.nih.gov/pubmed/16284649

12 Civitarese AE1, Carling S, Heilbronn LK, Hulver MH, Ukropcova B, Deutsch WA, Smith SR, Ravussin E; CALERIE Pennington Team. Calorie restriction increases muscle mitochondrial biogenesis in healthy humans. PLoS Med. 2007 Mar;4(3):e76.: http://www.ncbi.nlm.nih.gov/pubmed/17341128

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 8

Mitochondrial deficits appeared to be the mechanism causing insulin resistance and the associated age related diseases. The nitric oxide mechanism was activated to stimulate mitochondrial biogenesis ameliorating insulin resistance. What was still very puzzling was why would the human body allow the mitochondria to reduce their quantity resulting in free radicles leaking out of the mitochondria causing damage to the cells? Diabetics and people with insulin resistance and metabolic syndrome state they are tired all the time. Why would the human body not respond to the need for more energy and make more mitochondria through the nitric oxide or some other mechanism? There appeared to still be a missing link in understanding this nitric oxide signaling mechanism. There must still be something related to physical exercise that stimulates the nitric oxide / mitochondrial biogenesis mechanism beyond the simple increase in energy demand inherent in physical exercise. Then we found a paper published by researchers from the University of Buenos Aries where they measured the production of nitric oxide (via Nitric Oxide Synthase) related to voltage changes in the mitochondrial membranes13.

13 Valdez LB1, Zaobornyj T, Boveris A. (2006) Mitochondrial metabolic states and membrane potential modulate mtNOS activity. Biochim Biophys Acta. 2006 Mar;1757(3):166-72. Epub 2006 Mar 20. : http://www.ncbi.nlm.nih.gov/pubmed/16624252

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 9

The dependence on the mitochondrial metabolic state for the release of nitric oxide ( via mtNOS) may also explain why vigorous exercise to the point of breathlessness is more effective than walking and provide a possible explanation for whythe low hypoxic pressure deployed in the CVAC Process is possiblymore effective than EECP or Hyperbaric pressures.

This separation of energy demand from mitochondrial biogenesis made sense to us. Thousands of years ago individuals unable or unwilling to exercise would not have provide benefits to the survival of the tribe / family. They would have impaired the survival of the tribe or family. So the faster elimination of theburden of individual unproductive tribe / family members would have been a survival benefit for the tribe / family during times of scarcity of food or need to relocate quickly.

Our picture appeared complete. Like many concepts in theories in Physics and Electrical Engineering complicate equations describing complex interrelationships of feedback control systemsappeared to be boiling down to something very simple:The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 10

In a Nutshell, the mechanism supported by published science looked like this might be this simple:

Pressure changes during physical exercise were producing voltage changes on the surface of the mitochondria membranes due to the piezoelectric nature of the cell membranes1. These voltage changesunder the right metabolic environment were producing a release ofnitric oxide13 which was producing mitochondrial biogenesis4. Theincrease in mitochondria may be ameliorating the insulin resistance11 which in turn prevents the development of age relateddiseases9. This simplistic mechanism may explain the health benefits from exercise as well provide evidence for the hypotheses for the CVAC Process.

The study in mice conducted by Dr. Gutsaeva et.al14., when she was at Duke University, appeared to confirm and provide further evidence that the pressure changes inherent in short exposures tohigh altitude were producing mitochondrial biogenesis through thenitric oxide synthesis dependent mechanism. Of particular importance was her study measured mitochondria in the brain14. This study looked to be another very important signpost indicating we are on the right path on our Journey.

While this mitochondrial biogenesis perspective appeared very appealing with good data (sign posts) adequately connecting the roads , Carl Linton continually asserted that yes this mechanism is valid, however it is not a complete nor an adequate explanation for the CVAC process, exercise and allostasis.

Our Journey then proceeded to look for additional clues that could link published cellular and molecular biology studies to

14 Gutsaeva DR1, Carraway MS, Suliman HB, Demchenko IT, Shitara H, Yonekawa H,Piantadosi CA. (2008) Transient hypoxia stimulates mitochondrial biogenesis inbrain subcortex by a neuronal nitric oxide synthase-dependent mechanism. J Neurosci. 2008 Feb 27;28(9):2015-24. doi: 10.1523/JNEUROSCI.5654-07.2008.: http://www.ncbi.nlm.nih.gov/pubmed/18305236

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 11

the CVAC Process and physical exercise. In early 2012 we met a scientist by the name of Aubrey de Grey PhD. His theory involves7 mechanisms where by the mitochondrial concepts were one of the 7. In our meeting with him he was very complimentary of our published studies, our investigation path and methodology. We also began to learn about advanced gylcation end products (AGEs) and began to study the work of scientists involved with legitimate aging research. Many of those scientists told us AGEswere a dead end. They said, “More than a decade of research has shown nothing could be done about them.” This may be why Larry Ellison of the Ellison Foundation stopped funding Aging Research15 in December of 2013. However numerous published studies indicate AGEs are related to Diabetic complications16, Diabetic Neuropathy17,18, Diabetic Nephropathy19, Heart Disease20,

15 https://www.fightaging.org/archives/2013/12/ellison-medical-foundation-to-cease-funding-aging-research.php

16 Ahmed N. (2005) Advanced glycation endproducts--role in pathology of diabetic complications. Diabetes Res Clin Pract. 2005 Jan;67(1):3-21. : http://www.ncbi.nlm.nih.gov/pubmed/15620429

17 Huijberts MS, Schaper NC, Schalkwijk CG. (2008) Advanced glycation end products and diabetic foot disease. Diabetes Metab Res Rev. 2008 May-Jun;24 Suppl 1:S19-24.: http://www.ncbi.nlm.nih.gov/pubmed/18442180#

18 Sugimoto K, Yasujima M, Yagihashi S. (2008) Role of advanced glycation end products in diabetic neuropathy. Curr Pharm Des. 2008;14(10):953-61.: http://www.ncbi.nlm.nih.gov/pubmed/18473845

19 Thomas MC, Forbes JM, Cooper ME. (2005) Advanced glycation end products anddiabetic nephropathy. Am J Ther. 2005 Nov-Dec;12(6):562-72. : http://www.ncbi.nlm.nih.gov/pubmed/16280650

20 Zeinab Hegab, Stephen Gibbons, Ludwig Neyses, Mamas A. (2012) Role of advanced glycation end products in cardiovascular disease. World J Cardiol. Apr 26, 2012; 4(4): 90–102. : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342583/

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 12

Arthritis21, Skin Aging22,23, Alzheimer’s24,25 and Cancer26 . However,when we point out that exercise has been shown in one study27 to the lower the level of AGEs in the blood there is renewed excitement for this avenue of research. It is very important andinteresting to know that these aging researchers were not aware of this study.

21 DeGroot J1, Verzijl N, Wenting-van Wijk MJ, Jacobs KM, Van El B, Van Roermund PM, Bank RA, Bijlsma JW, TeKoppele JM, Lafeber FP.(2004) Accumulationof advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis. Arthritis Rheum. 2004 Apr;50(4):1207-15. : http://www.ncbi.nlm.nih.gov/pubmed/15077303

22 Wondrak GT1, Roberts MJ, Jacobson MK, Jacobson EL. (2002) Photosensitized growth inhibition of cultured human skin cells: mechanism and suppression of oxidative stress from solar irradiation of glycated proteins. J Invest Dermatol. 2002 Aug;119(2):489-98. : http://www.ncbi.nlm.nih.gov/pubmed/12190875

23 Gkogkolou P, Böhm M. (2012) Advanced glycation end products: Key players inskin aging? Dermatoendocrinol. 2012 Jul 1;4(3):259-70. : http://www.ncbi.nlm.nih.gov/pubmed/23467327

24Vitek MP1, Bhattacharya K, Glendening JM, Stopa E, Vlassara H, Bucala R, Manogue K, Cerami A. (1994) Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4766-70. : http://www.ncbi.nlm.nih.gov/pubmed/8197133 25Takeuchi M1, Kikuchi S, Sasaki N, Suzuki T, Watai T, Iwaki M, Bucala R, Yamagishi S. (2004) Involvement of advanced glycation end-products (AGEs) in Alzheimer's disease. Curr Alzheimer Res. 2004 Feb;1(1):39-46. : http://www.ncbi.nlm.nih.gov/pubmed/15975084

26 van Heijst JW1, Niessen HW, Hoekman K, Schalkwijk CG. (2005) Advanced glycation end products in human cancer tissues: detection of Nepsilon-(carboxymethyl)lysine and argpyrimidine. Ann N Y Acad Sci. 2005 Jun;1043:725-33.: http://www.ncbi.nlm.nih.gov/pubmed/16037299

27 Yoshikawa T1, Miyazaki A, Fujimoto S. Decrease in serum levels of advanced glycation end-products by short-term lifestyle modification in non-diabetic middle-aged females. Med Sci Monit. 2009 Jun;15(6):PH65-73.http://www.ncbi.nlm.nih.gov/pubmed/19478714

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 13

Was the reduction in AGEs in the blood a mitochondrial effect? Does keeping the free radical oxidants captive in the mitochondria, because there are more mitochondria, improve the gylcation efficiency reducing the production of waste products? Does this better glycation metabolize the waste products including dietary AGEs? Or is something else happening that could point us to travel down an additional road?

The research by Karen Herbst PhD MD on the CVAC Process in individuals with chronic pain and impaired lymphatic flow28 caused us to further investigate another avenue. While the improvements in mental scores was explainable by an increase in mitochondria the improvements in weight loss and pain in just 5 days of using the CVAC Process for just 40 minutes a day did not seem to fit well with the mitochondrial biogenesis concepts.

Additionally we began to receive numerous reports from about two dozen individuals and subjects in Dr. Linam’s formal pilot study who carried a diagnosis of diabetic neuropathy and were using theCVAC Process that their pain was being reduced and there was restoration of sensory function. These individuals would likely have been on medications that may inhibit mitochondrial biogenesis29,30.

28 Karen L Herbst, Thomas Rutledge. (2010) Pilot study: rapidly cycling hypobaric pressure improves pain after 5 days in adiposis dolorosa. J Pain Res. 2010; 3: 147–153.: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004643/

29 Robinson MM1, Hamilton KL, Miller BF. (2009) The interactions of some commonly consumed drugs with mitochondrial adaptations to exercise. J Appl Physiol (1985). 2009 Jul;107(1):8-16. : http://www.ncbi.nlm.nih.gov/pubmed/19423832

30 Mikus CR1, Boyle LJ, Borengasser SJ, Oberlin DJ, Naples SP, Fletcher J, Meers GM, Ruebel M, Laughlin MH, Dellsperger KC, Fadel PJ, Thyfault JP. (2013)Simvastatin impairs exercise training adaptations. J Am Coll Cardiol. 2013 Aug20;62(8):709-14. : http://www.ncbi.nlm.nih.gov/pubmed/23583255

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 14

Athletes were insistent that they were seeing improvements in recovery symptoms upon their first use of the CVAC process withintheir 20 minute or 40 minute session who had never taken a prior CVAC session; not the few or many more hours later that it wouldbe expected to take from a mitochondrial biogenesis effect based on the data from Dr. Gutsaeva14.

Finally, studies began to emerge linking sitting to an increase in diabetes and cancer independently from the amount of exercise31,32. People who exercised vigorously an hour a day had avery significant increased risk of developing diabetes if they had jobs that required them to sit most of the day. Based on thestudy by Dr. Gutsaeva14 the time constant for mitochondrial reduction after the nitric oxide mechanism was activated was too long to attribute these effects to a reduction in mitochondria.

So we began to look for other pathways and roads through the published scientific literature. We had observed immediate improvements in individuals with varicose veins and were aware that one way valves existed in veins. So on a hunch, we Googled: Lymphatic Valves

And saw these images:

31 Yates T1, Khunti K, Wilmot EG, Brady E, Webb D, Srinivasan B, Henson J, Talbot D, Davies MJ. (2012) Self-reported sitting time and markers of inflammation, insulin resistance, and adiposity. Am J Prev Med. 2012 Jan;42(1):1-7. : http://www.ncbi.nlm.nih.gov/pubmed/22176839

32 Katzmarzyk PT1, Church TS, Craig CL, Bouchard C. (2009) Sitting time and mortality from all causes, cardiovascular disease, and cancer. Med Sci Sports Exerc. 2009 May;41(5):998-1005. : http://www.ncbi.nlm.nih.gov/pubmed/19346988

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 15

So our research into published science involving mechanisms related to the CVAC process is now along another pathway and roads related to lymphatic stimulation that is likely inherent inphysical exercise as well as the CVAC Process and forming this concept:

The potential for the CVAC Process to be removing AGEs by way of lymphatic stimulation is now a keen focus of our validation research.

This discussion involves two pathways through the tremendous volumes of scientific literature that relate to allostasis, allostatic load and the balance between energy input and expenditure. As such it is not a complete explanation of the benefits of exercise, healthy fitness levels and the CVAC

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 16

process. We are certain there are more pathways to connect the roads of this science and many more roads to be created through original and confirming primary research.

One of the current additional pathways and roads we are exploringinvolves the removal of waste products and Tau from the brain by way of the glymphatic system33,34,35 for the purpose of improving brain function.

33 Iliff JJ1, Lee H, Yu M, Feng T, Logan J, Nedergaard M, Benveniste H. (2013)Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. J Clin Invest. 2013 Mar 1;123(3):1299-309. : http://www.ncbi.nlm.nih.gov/pubmed/23434588

34 Maiken Nedergaard. (2013) Garbage Truck of the Brain. Science. Jun 28, 2013; 340(6140): 1529–1530. : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749839/

35 Kress BT1, Iliff JJ, Xia M, Wang M, Wei HS, Zeppenfeld D, Xie L, Kang H, XuQ, Liew JA, Plog BA, Ding F, Deane R, Nedergaard M. (2014) Impairment of paravascular clearance pathways in the aging brain. Ann Neurol. 2014 Dec;76(6):845-61. : http://www.ncbi.nlm.nih.gov/pubmed/25204284

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 17

We invite you to join this Journey.

A particularly intriguing concept is at the junction of these twopathways that may have a profound effect on drug discovery, drug development and creating new roads of understanding:

o Do advanced glycation end products accumulate on the surfaces of the cell membranes insulating the effects of the voltage changes inhibiting nitric oxide release and inhibiting mitochondrial biogenesis as well as impairing the cells ability to communicate with other cells?

o Can mechanical (i.e. CVAC Process, exercise et.al.), biological or molecular mechanisms clean out the accumulation of these metabolic waste products (AGEs)?

o Is the CVAC Process more effective than traditional physical exercise because it removes the AGEs without an associated increase in production of the AGEs like is necessary in traditional physical exercise? If it ismore effective, what impact can that have on optimal health and disease free longevity?

o If energy availability is restored through mitochondrial biogenesis and AGEs cleaning / removal, will the lysosomes be able to have their function restored so they can regain their ability to remove AGEs?

o Do AGEs interfere with the function of the endoplasmic reticulum causing mis-folded proteins? Can reducing AGEs restore the proper function of the endoplasmic reticulum?

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 18

o Is a key factor in stem cell availability and effectiveness for the rejuvenation of the human body based on replacing old cells inflamed from oxidative stress damage and the accumulation of AGEs?

o Is the presence of AGEs in and on an old inflammed cellthat wants to die preventing a stem cell from replacingthe old cell? Does this inhibition in replacement contribute to the inflammation and progression seen in many of these diseases referenced in this discussion?

o We challenge you to develop an understanding and publish studies of the relationship of stem cell: mobilization, activation and biogenesis as it relates to physical exercise, nutrition and the CVAC Process.

The Scientific Journey to Investigate and Validate the CVAC Hypotheses Page 19