The evolving concept of physiologicalischemia training vs. ischemia preconditioning

Jun Ni,Hongjian Lu,Xiao Lu,Minghui Jiang,Qingyun Peng,Caili Ren,Jie Xiang, Chengyao Mei,Jianan Li,?

1Department of Rehabilitation Medicine,the Affiliated Hospital of Nantong University,Nantong,Jiangsu 226000,China;

2The Second People’s Hospital,Nantong,Jiangsu 226002,China;

3Department of Rehabilitation Medicine,the First Affiliated Hospital of Nanjing Medical University,Nanjing,Jiangsu 210029,China;

4Department of Cardiology,the Affiliated Hospital of Nantong University,Nantong,Jiangsu 226000,China.

The evolving concept of physiologicalischemia training vs. ischemia preconditioning

Jun Ni1,△,Hongjian Lu2,△,Xiao Lu3,Minghui Jiang4,?,Qingyun Peng4,Caili Ren3,Jie Xiang3, Chengyao Mei1,Jianan Li3,?

1Department of Rehabilitation Medicine,the Affiliated Hospital of Nantong University,Nantong,Jiangsu 226000,China;

2The Second People’s Hospital,Nantong,Jiangsu 226002,China;

3Department of Rehabilitation Medicine,the First Affiliated Hospital of Nanjing Medical University,Nanjing,Jiangsu 210029,China;

4Department of Cardiology,the Affiliated Hospital of Nantong University,Nantong,Jiangsu 226000,China.

Ischemic heartdiseases are the leading cause of death with increasing numbers ofpatients worldwide.Despite advancesin revascularization techniques,angiogenic therapiesremain highly attractive.Physiologicalischemia training,which isfirstproposed in ourlaboratory,refersto reversibleischemia training ofnormalskeletalmusclesby using a tourniquetorisometric contraction to cause physiologic ischemia forabout4 weeksforthe sake oftriggering molecularand cellularmechanisms to promote angiogenesis and formation ofcollateralvesselsand protectremote ischemia areas.Physiologicalischemia training therapy augmentsangiogenesis in the ischemic myocardium by inducing differentialexpression ofproteins involved in energy metabolism,cellmigration,protein folding,and generation.It upregulatestheexpressionsofvascularendothelialgrowth factor,and inducesangiogenesis,protectsthe myocardium when infarction occursby increasing circulating endothelialprogenitorcells and enhancing theirmigration,which is in accordancewith physicaltraining in heartdisease rehabilitation.These findingsmay lead to anew approach oftherapeutic angiogenesisforpatientswith ischemic heartdiseases.On the basisofthe promising resultsin animalstudies, studieswere also conducted in patientswith coronary artery disease withoutany adverse effect in vivo,indicating that physiologicalischemia training therapy is a safe,effective and non-invasive angiogenic approach forcardiovascular rehabilitation.Preconditioning isconsidered to bethe mostprotective intervention againstmyocardialischemia-reperfusion injury to date.Physiologicalischemia training is differentfrom preconditioning.This review summarizes the preclinicaland clinicaldata ofphysiologicalischemia training and its difference from preconditioning.

physiologicalischemia training,ischemia preconditioning,angiogenesis,vascularendothelialgrowth factor

Introduction

Ischemic cardiovasculardiseases including coronary artery disease(CAD)are the leading cause of death worldwide.Despite substantial advances in revascularization techniques such as surgicalrevascularization or percutaneous catheter intervention(PCI),angiogenic therapies remain the mainstay for the treatment ofischemic diseases[1].Myocardialischemia is important for the developmentof collateralcirculation[2].Intensive exercise training may facilitate collateral formation in CAD patients.However,the risk of high-intensity exercise in these patients is a major clinical concern. Therefore,it is necessary to explore approaches that can safely induce angiogenesis[1].

Physiological ischemia training(PIT)is a reversible ischemia training of normalskeletal muscles induced by tourniquetor isometric contraction to cause physiologic ischemia forabout1 month forthe sake ofdeveloping angiogenesisand collateralvesselsto protectthe ischemic area.PIT is usually applied after the occurrence of an ischemic vascular disease.It was first proposed in our laboratory as a noveland safe technique for cardiac rehabilitation[3-14].

Ischemic preconditioning(IPC)refers to the deliberate induction ofa series oftransientischemia and reperfusion before the lethal'index'ischemic eventoccurs.At present,there is a clearconsensus thatIPC is cardioprotective across all investigated animal species and is regarded as the most protective intervention against myocardial ischemia-reperfusion injury today[15-16]. Although IPC has been widely accepted,PIT remains a new conceptand is likely to be confused with IPC.

This article summarizes recent works of PIT in animals and humans,and discusses the potentialmechanism underlying PIT therapy that promotes collateral formation and the advantages of PIT.We also discuss the difference between PIT and IPC.

PIT therapy for myocardial infarction

Clinicalstudies have reported thatpatients who have had a myocardialinfarctmay have smaller infarcts[17]and a lower mortality rate if they had angina pectoris before[18].Itwas also demonstrated thatwell-developed collateral circulation may protect the residual viable myocardium and decrease the infarct size in patients with myocardialinfarction[19-20].

Lu et al.performed an animal experiment with pigs in vivo using a porcine model of chronic myocardial ischemia by implanting a balloon constrictorin the first obtuse marginal coronary artery[21].Itwas shown that high-intensity exercise could improve the formation of coronary collateralcirculation in pigs with myocardialischemia[21].Unfortunately,high-intensity exercise may be a significant risk for cardiovascular patients and has been limited for years due to the risk of triggering cardiovascular events[22,24].

Great progress has been made in PCI and the surgical revascularization field.However,there are still some patients with severe CAD who cannot sustain PCIorsurgicalrevascularization and they remain poor due to the lack ofeffective treatments.Therefore,safer approaches for inducing angiogenesis should be explored.PIT was firstproposed in our laboratory. Rabbitmodels ofcontrollable myocardialischemia are established by using an implanted waterballoon constrictor.Controllable coronary artery stenosisand reperfusion are induced by inflating and deflating the balloon by injecting and sucking outsterile water using a syringe, a processthatmimicsthe pathologic statusofstablemyocardialischemia[3-4,6].We treated these models with PIT for4 weeks.Stimulation wasapplied using a 1-m second square wavepulseapplied ata frequency of40 Hz,which was sufficientto cause the hind limb to produce 40%of itsmaximalforce withoutpain.The stimulation protocol wasrepeated for4 minutesin each session ata2-day intervalfor4 weeks.The experimenthas demonstrated that PIT induces the formation ofcollateralcirculation[3,6,10]. On the basis of these experiments,the 30 remaining rabbitswererandomly assigned into 3 groups:thepureischemia group thatunderwenta myocardialischemia only to imitate stable ischemia;the exercise training group that received PIT along with the same ischemic stimulation as the PIT group;the sham-operated group thatremained inactive.There were significantdifferences in infarctsize among the groups(P＜0.01)after 4 weeks of training. The findings suggestthatPIT for4 weeks can effectively decrease the myocardial infarct size,which is significantly correlated with heart function,myocardialblood flow and capillary density(CD).This is the first report thatdemonstrated the function of PIT in inducing collateralcirculation formation and protecting the myocardium when an infarction occurs.

PIT therapy for peripheral arterial disease

Vascular occlusion-induced peripheral arterial disease is a common disorder.With the rapid increase of the elderly population,the number of patients with peripheral arterial disease continues to increase.To date,critical limb ischemia in peripheral arterialdisease has become a greatchallenge in medicaltherapy.

A numberofstudies have reported thatangiogenesis is an effective method to improve collateral-dependent blood flow to distal tissues at risk of ischemia,and demonstrated that exercise can induce angiogenesis in the peripheralcirculation[25].Most studies focused theirattention of electricalstimulation oraerobic exercise primarily on the localischemic muscle.In fact,upregulation of angiogenic factors should notbe limited to a localsite.Remote expression ofangiogenic factors induced by exercise and ischemia has been reported[26-27].Liu etal.reported the up-regulation ofvascularendothelialgrowth factor(VEGF)in serum and skeletalmuscles after myocardialischemia[28].

As localexercise increases cytokines and leucocytes in the circulation or non-exercised tissues,itmay promote angiogenesis in the distalpathological ischemic site via a remote effect of the angiogenic factors. Therefore,a study was designed to testthe hypothesis that local exercise of a normal limb could promote angiogenesis in the pathologically ischemic limb. Animalexperiments were performed in a rabbitmodel by chronically ligating the femoral artery of the left hindlimb,and implanting an electrode onto the sciatic nerve of the right hindlimb[10].The animals were then randomly assigned to 4 groups,including the Lig-N group without receiving electrical stimulation,the Lig-High group receiving high-intensity electrical stimulation on the right hindlimb,the Lig-Low group receiving low-intensity electrical stimulation on the righthindlimb),and the Double-Lig-High group receiving both ligation of the left femoral artery and highintensity electrical stimulation on the right hindlimb. In the Lig-High and Double-Lig-High groups,the high-intensity electrical stimulation was applied at a frequency of 40 Hz for a 1-m second duration,and the maximal force reached a plateau of 13-16 N at a currentof2.5 mA.In the Lig-Low group,low-intensity electricalstimulation was defined as 0.3 mA(40 Hz for 1 m second)to cause minimalmacroscopic muscle contraction.The electrical stimulation procedure included an episode of 5-minute stimulation and a 5-minute rest, 8 times daily for4 weeks.Upon gross examination with angiography,the number of collaterals was significantly higher in the Lig-High group compared with the three groups(P＜0.01).Blood flow,capillary density and VEGF mRNA levels in the gastrocnemius muscle in the Lig-High group were also noticeably higher than the other three groups(P＜0.01).The value of VEGF protein levels in the Lig-High group was significantly higher than that of the other three groups(P＜0.01). The findingsdemonstrated thatPIT in normallimbswith high intensity electricalstimulation may significantly increase collateralblood flow and capillary supply in the pathologicalischemia site,indicating thatphysiologicalischemic exercise training in the normallimb for 4 weeks facilitated angiogenesis and up-regulated VEGF expression in the pathologicalischemic limb[9-10].

Mechanisms underlying PIT therapy

Molecule mechanisms

Although the underlying mechanism responsible for the effectof PIT remains notclearly understood,some evidence may provide meaningful insights.Firstly, ischemic exercise of the normalskeletal muscles elevates circulating VEGF binding to the site with pathological ischemia.Remote expression of VEGF has been demonstrated in the skeletal muscles induced by 4-week stenosis of the coronary artery[28].Secondly, apart from VEGF synthesis in the muscle,circulating T-cells[29]and monocytes[30]provide another source of VEGF.Our study demonstrated that PIT augmented angiogenesis in the ischemic myocardium through differential expression of proteins involved in energy metabolism,cellmigration,protein folding,and generation[31].Logically,remote expression of angiogenic factors implies potentialremote angiogenesis.

Coronary collateralsmay develop in response to intermittentbrief myocardial ischemia in humans,and that collaterals may be preserved and could immediately function when a coronary artery is acutely occluded. This notion has been supported by some researchers who found that coronary collateral circulation could extend the"golden time"before coronary reperfusion when infarction occurred.Well-developed collaterals were found to decrease the infarctsize of patients who had experienced infarction[32-33].Only useful,efficient, or functionalcollateralflow could preventmyocardial ischemia.Our study supports these findings.

Treadmilltestshowed thatexercise training increased VEGF levels by 310%in patients with peripheralarterialdisease,where the presence of ischemia was documented as an increase in venous lactate levels and complaint of ischemic leg pain.With an increase in exercise intensity,lactate accumulated in the muscle and the concentration of lactate elevated significantly. Atbaseline and immediately after exercise,venous lactate wasmeasured to confirm ischemia and hypoxia during high-intensity electricalstimulation exercise training. Rabbits in the low-intensity group were notsubjected to ischemic stimulation.The absence of ischemia was shown by no change in venous lactate levels.The levels of VEGF mRNA and protein in the leftgastrocnemius muscle were allup-regulated in the high-intensity group compared with those in the low-intensity group.This is due to the induction of ischemia by high-intensity exercise in the Lig-High group[10].We used high frequency (40 Hz)to produce intense muscle contractions.Highintensity static muscle contraction is importantto perform resistive exercises[10].

The higher intensity of resistance will accountfor the larger proportion of static muscle contractions in resistive exercises.According to the previous results[9-10], high-intensity resistance exercise promoted angiogenesis in remote ischemic tissues.However,high-intensity exercise has been limited as a treatment for patientswith cardiovasculardisease because ofthe cardiovascular risk[7].This finding may lead to a potential new approach of applying PIT of skeletal muscles in the treatment of patients with ischemic diseases.

Cellular mechanisms

The notion that PIT could increase systemic endothelial progenitor cells(EPCs)has been supported by the literature.A 12-week period of regular physical exercise resulted in a 2.9-fold increase in circulating EPCs,and therefore promoted cardiovascularhealth[34]. However,another study[5]showed that PIT could also increase circulating EPCs,which is in accordance with physicaltraining in heartdisease rehabilitation.

Itwas also found thatexercise induced a highly significant enhancement of migratory capacity in all groups[34].Wan C investigated the relationship between PIT and the function of EPCs.PIT,like circulating EPCs,also increases both the quantity of migrating EPCs and the migratory activity of EPCs,which is consistentwith the situation in physicaltraining.In addition,PIT improves capillary density in the ischemic cardiac area,which is in accordance with the circulating EPCs and EPC migration activity,and hence might be applied as a promising new approach in cardiac rehabilitation.The statistics showed that circulating EPCs were positively correlated with capillary density in the ischemic cardiac area.This may be due to homing of EPCs.When EPCs homed to the injured tissue, they could promote angiogenesis and then improve local microenvironment.Meanwhile,the improved microenvironment in turn could promote homing and functioning of EPCs.Our study showed thatthe homing of EPCs wasinvolved in the effectof PIT on angiogenesis in the remote ischemic cardiac area,during which the quantity and activity of EPCs were shown to be the key factors.

Clinical studies

A recentstudy was designed by Lin S et al.[12]has confirmed that PIT in patients with CAD could increase coronary collateral flow during acute vessel occlusion,which was significantly differentfrom controlocclusion.A randomized controlled study was performed to determine whether isometric exercise could increase collateralflow in the remote ischemic myocardium in an acute coronary occlusion model in 65 patients with one-vessel CAD.The patients were randomly assigned to either the isometric exercise group or non-exercise group.Patients in the exercise group performed isometric handgrip exercises(50%maximal voluntary contraction)during 1-minute coronary balloon occlusion,while patients in the non-exercise group remained sedentary.Collateralflow index,heart rate,systolic blood pressure and diastolic blood pressure were determined prior to and 1 minute after coronary occlusion.In the exercise group,the collateralflow index after and before coronary occlusion was significantly higher than that of the non-exercise group (P＜0.01),and so were heartrate,systolic and diastolic blood pressure(P＜0.01).

Evolving concept of preconditioning

Currently,there is a clear consensus that IPC is cardioprotective across allanimalspecies investigated and is the mostprotective intervention againstmyocardial ischemia-reperfusion injury[15].Remote ischemic conditioning was firstdiscovered to offer protection within one organ[35],gaining interestas a potential method to induce resistance against ischemia reperfusion injury in a variety of clinical settings.Itis an approach of conditioning in which not the target organ,such as the heart,butinstead a more accessible tissue is submitted to a conditioning stimulus.Remote preconditioning has also been currently found to offer protection against ischemia reperfusion injury when a different organ, oreven the skeletalmuscle tissue is used forconditioning[36].However,it is a matter of fact that remote ischemic conditioning reduces the incidence of periproceduralmyocardial infarction and the release of troponin.There is no evidence thatremote ischemic conditioning reduces ischemic event-related mortality,nor does itreduce major adverse cardiovascular events.

The conceptof PIT is differentwith preconditioning, which renders the organ resistantto a more prolonged episode of ischemia.Remote ischemic preconditioning applies the precedent ischemic stimulus to a distant site from the organ or tissue that is afterward exposed to ischemia injury.PIT needs to train for severalweeks while adaptation of IPC or remote ischemic preconditioning only needs several times.The underlying mechanisms of PIT and remote ischemic preconditioning are very complex and notyetfully defined.Ithas been hypothesized thatremote ischemic preconditioning predominantly involves systemic multifactorial antiinflammatory,neuronal and humoral signaling pathways,which are likely to interact with each other[37]. Remote ischemic preconditioning has been described to reduce ischemia-reperfusion injury in various animal models.Remote effect of PIT may facilitate coronary collateralformation ofthe myocardium by repeated episodes of short-term skeletalmuscle ischemia after the occurrence ofan ischemic event.PIT therapy augments angiogenesis in the ischemic myocardium by inducingdifferential expression of proteins involved in energy metabolism,cellmigration,protein folding,and generation[14,31].It upregulates the expressions of VEGF and VEGF mRNA,induces angiogenesis,protects the myocardium when infarction occurs by increasing circulating EPCs and enhancing migration as well as migratory activity of EPCs[3,5,8,10].Further studies are needed to understand the differentmechanisms.

The cardioprotective effect of short-term skeletal muscle ischemia has been previously evaluated in experimental and clinical studies and the beneficial effect on the ventricular myocardium is not specific for a particular species[38].Physiologic ischemia is reversible noninvasive ischemia of normal skeletal muscles caused by tourniquet or isometric contraction, and induces the developmentofcollateralcirculation in the myocardium.The beneficial effect of repeated ischemic training arises from collateral formation rather than from tissue adaptation.

Conclusion

Our research has demonstrated that physiologic ischemic training induces collateralcirculation formation and protects the myocardium from complete occlusion ofa coronary artery and peripheralarterialdisease. These findings may serve as a theoretic basis for furtherresearch and a new way of thinking for rehabilitation of ischemic disease.

Acknowledgement

This work was supported by grants from Nantong City(No.BK2014040 to Jun Niand No.HS2012057 to Hongjian Lu),and from Chinese National Science Fund of China(No.81070181 to Xiao Lu).

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△These authors contribute equally to the work.

?Corresponding author:Dr.Jianan Li,the First Affiliated Hospital of Nanjing Medical University,Nanjing,Jiangsu 210029,China;Tel/fax: 13705161766/+86-025-84437892,E-mail:lijianan@carm.org.cn; Dr.Minghui Jiang,Department of Cardiology,the Affiliated Hospital of Nantong University,Nantong,Jiangsu 226000,China,Tel/fax:

13906290505/+86-025-84437892,E-mail:jiangminhui@medmail. com.cn.

R541.4,Document code:A

These authors reported no conflict of interests.

Received 23 October 2014,Revised 24 January 2015,Accepted 22 May 2015,Epub 10 September 2015

?2015 by the Journal of Biomedical Research.All rights reserved.

10.7555/JBR.29.20140142