Wound Healing Angiogenesis:Innovations and Challenges in Acuteand Chronic Wound Healing

Tatiana N. Demidova-Rice,1 Jennifer T. Durham,2 and Ira M. Herman2,*1Department of Radiation Oncology, Edwin L. Steele Laboratory for Tumor Biology, Massachusetts General Hospital,

Boston, MA 02114.2Department of Molecular Physiology and Pharmacology and the Center for Innovations in Wound Healing Research,

Tufts University School of Medicine and Graduate Program in Cellular and Molecular Physiology,

Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA 02111.

Background: Formation of new blood vessels, by either angiogenesis or vascu-logenesis, is critical for normal wound healing. Major processes in neovascu-larization include (i) growth-promoting or survival factors, (ii) proteolyticenzymes, (iii) activators of multiple differentiated and progenitor cell types, and(iv) permissible microenvironments. A central aim of wound healing research isto ‘‘convert’’ chronic, disease-impaired wounds into those that will heal.The problem: Reduced ability to re-establish a blood supply to the injury sitecan ultimately lead to wound chronicity.Basic/Clinical Science Advances: (1) Human fetal endothelial progenitor cellscan stimulate wound revascularization and repair following injury, as demon-strated in a novel mouse model of diabetic ischemic healing. (2) Advances inbioengineering reveal exciting alternatives by which wound repair may be fa-cilitated via the creation of vascularized microfluidic networks within organconstructs created ex vivo for wound implantation. (3) A ‘‘personalized’’ ap-proach to regenerative medicine may be enabled by the identification of proteincomponents present within individual wound beds, both chronic and acute.Clinical Care Relevance: Despite the development of numerous therapies, im-paired angiogenesis and wound chronicity remain significant healthcare prob-lems. As such, innovations in enhancing wound revascularization would lead tosignificant advances in wound healing therapeutics and patient care.Conclusion: Insights into endothelial progenitor cell biology together with de-velopments in the field of tissue engineering and molecular diagnostics shouldnot only further advance our understanding of the molecular mechanismsregulating wound repair but also offer innovative solutions to promote thehealing of chronic and acute wounds in vivo.

BACKGROUNDAngiogenesis, the formation of

new blood vessels from the preexist-ing vasculature, is indispensablefor successful wound healing. Post-injury, microvascular endothelialcells (ECs) that line the inner surfaceof blood vessels are activated by lowoxygen tension/hypoxia and proan-giogenic factors, including vascular

endothelial growth factor (VEGF). Inturn, ECs degrade their surroundingextracellular matrix, begin migra-tion and cell division (proliferation),and then reestablish cell–cell con-tacts, forming new capillaries.1 An-other critical process required forwound revascularization is vasculo-genesis, which relies on the mobili-zation of endothelial progenitor cells

Ira M. Herman

Submitted for publication June 27, 2011.

*Correspondence: Department of Molecular

Physiology and Pharmacology and the Center for

Innovations in Wound Healing Research, Tufts

University School of Medicine and Graduate

Program in Cellular and Molecular Physiology,

Sackler School of Graduate Biomedical Sciences,

Tufts University, Boston, MA 02111 (e-mail:

ira.herman@tufts.edu)

Abbreviationsand Acronyms

EC = endothelial cell

EPC = endothelial progenitorcell

FPC = fetal progenitor cells

FPC133 + = CD133-positivefetal progenitor cells

VEGF = vascular endothelialgrowth factor

j 17ADVANCES IN WOUND CARE, VOLUME 1, NUMBER 1Copyright ª 2012 by Mary Ann Liebert, Inc. DOI: 10.1089/wound.2011.0308

(EPCs) from the bone marrow to areas of regenerat-ing or healing tissues. EPCs (both embryonic andadult) possess several stem-cell like surface markers,including the cell surface glycoprotein CD133, andcan differentiate into several cell types.2 Althoughstem cell applications have proven beneficial in car-diovascular settings,3 EPCs’ efficacy in fosteringdiabetic wound healing remains problematic, be-cause diabetic patients possess both diminished anddysfunctional EPCs4,5 as well as unfavorable woundmicroenvironments. As a need for molecular andcellular therapeutics remains evident, alternativepathways are being explored, including the devel-opment of implantable bioengineered microvascularnetworks that would enhance wound bed perfusion.6

Ultimately, understanding patient-specific meta-bolic and molecular profiles7 should provide potentdiagnostic tools, advancing the field toward a per-sonalized regenerative medicine-based approach.

CLINICAL PROBLEM ADDRESSED

Cells and tissues are inherently dependent on thevasculature to supply critical nutrients and oxygenin exchange for metabolites. Impaired wound revas-cularization can impede healing, just as insufficientperfusion is a hallmark of chronic wounds. Severalapproaches including delivery of proangiogenicgrowth factors8 have been employed to enhancewound healing; however, current treatments remain

technologically or biologically hampered.9 Thus, thechallenge persists to create innovative and effectivetherapeutics for acute and chronic wound repair.

BASIC SCIENCE CONTEXT

A growing body of evidence has advanced ourunderstanding of the mechanisms leading to non-healing, chronic wounds.10 These include, butare not limited to, reduced bioavailability of growthfactors and receptors, abnormal production/modification of matrix proteins, diminished prolifer-ative capacity of resident cells, and insufficientor impaired wound perfusion (Fig. 1). The latterstems from EC dysfunction as well as impaired re-cruitment of EPCs to the injury site.11 One method tocircumvent this issue is the application of exogenousadult or fetal progenitor cells (FPCs) to chronicwound beds.12 Commonly expressing the EPC sur-face markers CD133, CD34, and VEGF receptor-2(KDR), these cells can differentiate into both vascularendothelial and perivascular (mural) cells.2 In addi-tion, CD133-positive cells generate proangiogenic

TARGET ARTICLES

1. Barcelos LS, Duplaa C, Krankel N, GraianiG, Invernici G, Katare R, Siragusa M, Meloni M,Campesi I, Monica M, Simm A, Campagnolo P,Mangialardi G, Stevanato L, Alessandri G,Emanueli C, and Madeddu P. Human CD133 +progenitor cells promote the healing of diabeticischemic ulcers by paracrine stimulation ofangiogenesis and activation of wnt signaling.Circ Res 2009; 104: 1095.

2. Borenstein JT, Megley K, Wall K, Pritch-ard EM, Troung D, Kaplan DL, Tao SL, andHerman IM: Tissue equivalents based on cell-seeded biodegradable microfluidic constructs.Materials 2010; 3: 1833.

3. Eming SA, Koch M, Krieger A, BrachvogelB, Kreft S, Bruckner-Tuderman L, Krieg T,Shannon JD, and Fox JW: Differential proteomicanalysis distinguishes tissue repair biomarkersignatures in wound exudates obtained fromnormal healing and chronic wounds. J ProteomeRes 2010; 9: 4758.

FIG. 1. Chronic wounds are often characterized by hyperglycemia, per-sistent inflammation, and growth factor and cytokine receptor deficiencies,which lead to impaired progenitor cell recruitment and angiogenesis anddelayed epithelialization. Despite the progress achieved in the developmentof wound healing therapeutics, significant problems persist. Novel woundhealing approaches that aim to include adult or embryonic progenitor cellsor vascularized skin grafts seem promising, yet these methods will requirefurther preclinical and clinical evaluation before normalization of thechronic wound microenvironment fosters the restoration of wound healing.

18 DEMIDOVA-RICE ET AL.

soluble mediators, such as VEGF,2 and areable to promote revascularization follow-ing pathologic injury, including ischemicheart disease.13 Although fetal or adultendothelial progenitor cells may poten-tially offer benefits for chronic wound suf-ferers, further study will be needed toensure safety and efficacy. Also, alternativestrategies for wound revascularizationare being investigated including the useof bioengineered vascularized scaffolds.6

Linked to this, successful wound revascu-larization will require a permissive micro-environment, which could be adjusted in apatient-specific manner based on woundconditions determined using modern pro-teomics or point-of-care diagnostics.7

EXPERIMENTAL MODELOR MATERIAL—ADVANTAGESAND LIMITATIONSTreatment of ischemic woundsin mice using human FPCs

Isolating human fetal aortic progenitorcells represents a considerable challenge,technically and ethically,12,14 requiringrigorous cell selection, recovery, and prop-agation. For example, Barcelos and co-authors2,12 used EC-specific anti-CD31 toeliminate differentiated ECs and anti-CD133 antibody to enrich for progenitorcells. To evaluate the proangiogenic potential ofthese cells, a newly established mouse model of is-chemic wound healing was used, which relies uponpharmacologic induction of diabetes, followed by is-chemic insult. This model recapitulates both thehyperglycemia accompanying diabetes as well as theischemia-reperfusion issues characteristic of chronicwounds. Therefore, this is an attractive model to testa variety of cellular and molecular-based healingmodalities, including progenitor cells, under a moreclinically relevant setting.

Creation of prevascularized microfluidicnetworks in biodegradable scaffolds

Microvascular networks with dimensions andfeatures resembling the human dermal microvas-culature are being constructed using biocompatiblebiomaterials.6 Such silk scaffolds can be seededwith micro- or macrovascular-derived ECs andperfused ex vivo. Moreover, these cellularized mi-crofluidic networks can be layered using otherdermal compartment cells or overlaid with epithe-lial cellular components. Clearly, further work will

be necessary to ensure optimal host engraftmentin vivo of such vascularized ‘‘organ equivalents.’’

Analysis of protein profiles of acuteand chronic wounds

Wound exudates were noninvasively collectedfrom either acute or chronic (venous) wounds.7 Re-presentative samples were subjected to proteinidentification analysis using mass spectrometry.The subsequently identified proteins, not previ-ously linked to wound healing or its impairment,have been further characterized using immunohis-tochemistry. Challenges include both the intensiveeffort of data analysis as hundreds of proteins areidentified in this manner, and a likely omission ofpotentially important yet underrepresented targetsundetectable using mass spectrometry techniques.

DISCUSSION OF FINDINGSAND RELEVANT LITERATURE

During normal wound healing, EPCs are effec-tively recruited to the remodeling microcircula-tion, thus leading to wound revascularization

TAKE-HOME MESSAGEBasic science advances

� Human embryo–derived CD133 + progenitor cells promote both angio-genesis and wound healing in diabetic ischemic ulcers in mice.

� The wound healing effects of live human embryo–derived CD133 +progenitor cells can be replicated in the absence of cells by the use ofcell-free media containing soluble factors, such as VEGF produced byCD133 + cells (conditioned media).

� Angiogenic stimulation is achieved via soluble factor–mediated increasein endothelial survival, proliferation, and migration.

� Nonimmunogenic silk-based channels can support survival and growth ofmicrovascular ECs.

� The wound exudates derived from acute and chronic wounds have dif-ferent protein composition.

� The molecules that were exclusively found in healing wounds includeseveral types of collagens, thrombin, and heparan sulfate proteogly-can—all critical for wound revascularization.

Clinical science advances

� Proangiogenic embryo-derived CD133 + progenitor cells or the cell-conditioned media have robust wound healing potential in vivo.

� Microfluidic devices might provide a tool for revascularization of chronicwounds and stimulation of their healing.

Relevance to clinical care

� Noninvasive probing of wound microenvironment using modern pro-teomics together with CD133 + cells and vascularized skin grafts mayprovide useful tools for the personalized medicine.

ANGIOGENESIS OF WOUND HEALING 19

and timely healing. This response is likely to bedampened in diabetic ulceration. Indeed, it hasbeen recently demonstrated that EPCs from nor-mal but not diabetic patients contribute to postis-chemic revascularization.4 Diabetic EPCs are bothlower in number5 and dysfunctional, displaying ashift toward a proinflammatory phenotype.15 Nor-mal adult and/or FPCs (i) can differentiate intoseveral cell types and (ii) have stimulatory effectson biological processes and are therefore likely tobe beneficial for chronic wound patients and thosewith impaired perfusion. However, successful uti-lization of stem cells for chronic wound healing stillneeds further development and optimization. In arecent study, fetal CD133-positive cells (FPC133 + ),isolated from human fetal aortas and expanded aspreviously described,2 were used to stimulate is-chemic wound healing in diabetic mice using acollagen-based delivery system.12 Both FPC133 +and FPC133 + -conditioned media accelerate ratesof wound closure, increase EC proliferation, andpromote wound revascularization. Strikingly, aminimal number of vascular-associated FPC133 +are observed in wounds at 3 days postinjury/trans-plantation, yet the proangiogenic effects persistedfor 7 days, suggesting an indirect (paracrine)mechanism that sustains FPC functionality post-injury. In fact, several soluble mediators, includingVEGF, interleukin-6, interleukin-8, monocytechemoattractant protein, and granulocyte-colonystimulating factor, have been validated as producedby FPC133 + .12 This observation provides an im-portant insight into the mechanisms of stem cell–mediated repair. Of note, CD133 + cells are found inseveral human cancers16; therefore, their deliveryto wounds might be undesirable. On the other hand,proangiogenic cytokines generated by CD133 +cells provides a means to replace live progenitorcells with their conditioned media. Although cell-freepreparations containing bioactive molecules demon-strate wound healing potential in animal models,12 amajor obstacle to this therapy is the insufficientgrowth factor receptor density and responsiveness ofcells residing within the wound bed, as is often thecase among chronic wound sufferers.17 Alternativestrategies include modification of patient-specificEPCs, themselves, through inhibition of certain cellsignaling pathways to increase reparative func-tion.18 All together, progenitor cell therapy and/orappropriate modifications could prove beneficial topromote a chronic healing phenotype to an acute re-sponse. Additional work is needed to delineate celltype, modification protocol, and mode of delivery.

Currently, no consensus exists regarding thesafety and efficacy of exogenous/endogenous stem

cell applications for wound healing. As an alter-native, the delivery of biomaterial scaffolds to fos-ter host-specific recovery has been suggested. Forexample, silk fibroin, prepared as previously de-scribed,6 is routinely used for adult progenitor cellgrowth and differentiation.19 Moreover, such bio-materials can be fabricated into complex designsthat mimic vascular branching patterns in vivo. Inturn, the silk-based scaffolds can be populatedwith competent differentiated ECs, with fluid flowas a patterning guide. Described by Borenstein andcoauthors,6 biodegradable microfluidic constructswere seeded with human microvascular ECs,which remained viable and retained their cell sur-face markers for over a week. Additional modifi-cations include coculturing with perivascular cellsas well as inclusion of a keratinocyte cell layerto create tissue equivalents for implantation.Although the successful implantation of suchmicrofluidic devices into a wound bed in vivo hasnot been yet described, beneficial effects on woundrevascularization and healing are anticipated.

Successful wound revascularization is largelydependent on a permissive wound microenviron-ment. However, the identification and standardi-zation of the molecular profiles of the individualwounds remains challenging. Recently, a conceptof ‘‘wound bar coding’’ was proposed20 to provide aclassification scheme based on gene expressionpatterning by resident or peripheral wound bedcells. The levels of gene activity can be assessedusing modern RNA microarray technologies andpresented in a ‘‘heat map’’ style, depicting both up-and downregulated genes. This strategy providespotent information, which may both guide wounddebridement and lead to appropriate selection oftreatment strategies based on individual patientneeds (personalized medicine). However, severalpotential drawbacks include the requirement ofmultiple biopsies from numerous locations andthe inability to assay for specific proteins within awound. Therefore, a more conducive option mightbe the noninvasive probing of the wound environ-ment from wound exudates as was reported byEming and coauthors.7 This test not only enablesrepetitive monitoring of gene expression patternsbut also determines protein composition of woundmicroenvironments and thus might provide pow-erful predictions of the actual conditions within thewound. A recent study of comparative proteomics ofacute and chronic wounds using this approach7 hasdemonstrated that the nonhealing ulcers have de-creased amount of several extracellular matrixcomponents, namely, heparan sulfate proteoglycanand collagen type I, both of which are critical for

20 DEMIDOVA-RICE ET AL.

normal wound revascularization.21,22 Moreover,this study confirms a previously established andsignificant increase in production of multiple prote-ases, which are detrimental for both wound extra-cellular matrix components and proangiogenicgrowth factors.23 In addition, it identifies speciesthat are exclusively present in acute healing, such asthe serine protease thrombin and the antimicrobialdermicidin. Finally, these authors report the identi-fication of certain proteins, which have never beenpreviously associated with wound healing, includingthe matrix molecule olfactomedin-4. Together, theseresults shed light on how the normalization of woundmicroenvironment in a patient-specific manner,which in turn could be directed by microarray20 andproteomic analysis,7 would culminate in the promo-tion of wound revascularization or healing.

INNOVATION

In recent decades, embryonic and adult endo-thelial progenitor cells have been proposed to havetherapeutic potential. However, it remains to beshown whether these cells are applicable fortreatment of ischemic diabetic wounds. The work ofBarcelos and coauthors12 demonstrates that, infact, FPC133 + have proangiogenic properties inthis model and thus might be a useful tool fortreatment of ischemic wounds in diabetic patients.Further, endothelialized microvascular networksbased on degradable silk scaffolds6 may provide avaluable platform for future development of vas-cularized biomaterials. Finally, identification ofprotein components within wound microenviron-ments advances the field reach of the developmentof personalized approaches to wound care.

CAUTION, CLINICAL REMARKS,AND RECOMMENDATIONS

Althoughundifferentiated human-derivedFPC133+could be considered a helpful tool for chronic woundrevascularization, significant risks to the patients,including risk of cancer development and infec-

tious disease transmission, exist. Additional re-search remains to clarify progenitor cell biologicalprocesses and prevent undesirable effects of cell-based therapies.

� Progenitor cells can be differentiated in vitroand delivered into the wounds within bioma-terial-based scaffolds, thus reducing the riskof undesirable cell differentiation in vivo.

� Combination of RNA- and protein-based di-agnostic tools could be used to direct suc-cessful wound revascularization and healing.

FUTURE DEVELOPMENT OF INTEREST

The use of adult and/or embryonic endothelialprogenitor cells in combination with biocompatibleartificial microvascular networks could provide auseful tool for revascularization and enhancement ofwound healing. Optimized protocols designed to ob-tain and expand patient-compatible stem cells shouldbe devised and would continue to add to the regimenof personalized medicine. Additional studies usingnovel animal models will determine the cellular andmolecular mechanisms that underlie acute andchronic healing and provide a better understandingof the basic biology underlying stem cell application,which would demonstrate both the safety and effi-cacy of this treatment for the clinic. Insightful crea-tion of therapeutic interventions as well as powerfulpredictive tools would positively affect the outcomesfor managing and healing chronic wounds. Ulti-mately, these approaches should be based on per-sonalized approaches to wound diagnosis andinnovative treatment modalities in efforts to decreasethe chronic wound burden of the population.

ACKNOWLEDGEMENTS AND FUNDINGSOURCES

This work was funded by NIH grants (EY15125and EY19533).

AUTHOR DISCLOSURE AND GHOSTWRITING

I.M. Herman is a consultant for Healthpoint, Inc.

REFERENCES

1. Singer AJ and Clark RA: Cutaneous wound heal-ing. N Engl J Med 1999; 341: 738.

2. Invernici G, Emanueli C, Madeddu P, Cristini S,Gadau S, Benetti A, et al.: Human fetal aortacontains vascular progenitor cells capable ofinducing vasculogenesis, angiogenesis, andmyogenesis in vitro and in a murine modelof peripheral ischemia. Am J Pathol 2007; 170:1879.

3. Asahara T, Murohara T, Sullivan A, Silver M, vander Zee R, Li T, et al.: Isolation of putative pro-genitor endothelial cells for angiogenesis. Science1997; 275: 964.

4. Caballero S, Sengupta N, Afzal A, Chang KH, LiCalzi S, Guberski DL, et al.: Ischemic vasculardamage can be repaired by healthy, but not dia-betic, endothelial progenitor cells. Diabetes 2007;56: 960.

5. Brunner S, Hoellerl F, Schmid-Kubista KE, Zeiler F,Schernthaner G, Binder S, et al.: Circulating an-giopoietic cells and diabetic retinopathy in T2DMpatients with and without macrovascular disease.Invest Ophthalmol Vis Sci 2011; 52: 4655.

6. Borenstein JT, Megley K, Wall K, Pritchard EM,Truong D, Kaplan DL, et al.: Tissue equivalentsbased on cell-seeded biodegradable microfluidicconstructs. Materials 2010; 3: 1833.

ANGIOGENESIS OF WOUND HEALING 21

7. Eming SA, Koch M, Krieger A, Brachvogel B, KreftS, Bruckner-Tuderman L, et al.: Differential pro-teomic analysis distinguishes tissue repair bio-marker signatures in wound exudates obtainedfrom normal healing and chronic wounds. J Pro-teome Res 2010; 9: 4758.

8. Leahy PJ and Lawrence WT: Biologic enhance-ment of wound healing. Clin Plast Surg 2007;34: 659.

9. Schultz GS, Davidson JM, Kirsner RS, Bornstein P,and Herman IM: Dynamic reciprocity in the woundmicroenvironment. Wound Repair Regen 2011;19: 134.

10. Falanga V: Wound healing and its impairment inthe diabetic foot. Lancet 2005; 366: 1736.

11. Liu ZJ and Velazquez OC: Hyperoxia, endothelialprogenitor cell mobilization, and diabetic woundhealing. Antioxid Redox Signal 2008; 10: 1869.

12. Barcelos LS, Duplaa C, Krankel N, Graiani G, In-vernici G, Katare R, et al.: Human CD133 + pro-genitor cells promote the healing of diabeticischemic ulcers by paracrine stimulation of an-giogenesis and activation of wnt signaling. CircRes 2009; 104: 1095.

13. Adler DS, Lazarus H, Nair R, Goldberg JL, GrecoNJ, Lassar T, et al.: Safety and efficacy of bonemarrow-derived autologous CD133 + stem celltherapy. Front Biosci (Elite Ed) 2011; 3: 506.

14. Gucciardo L, Lories R, Ochsenbein-Kolble N, Done’E, Zwijsen A, and Deprest J: Fetal mesenchymalstem cells: isolation, properties and potential usein perinatology and regenerative medicine. BJOG2009; 116: 166.

15. Loomans CJ, van Haperen R, Duijs JM, VerseydenC, de Crom R, Leenen PJ, et al.: Differentiation ofbone marrow-derived endothelial progenitor cellsis shifted into a proinflammatory phenotype byhyperglycemia. Mol Med 2009; 15: 152.

16. Monzani E, Facchetti F, Galmozzi E, Corsini E,Benetti A, Cavazzin C, et al.: Melanoma containsCD133 and ABCG2 positive cells with enhancedtumourigenic potential. Eur J Cancer 2007; 43: 935.

17. Krishnan ST, Quattrini C, Jeziorska M, Malik RA,and Rayman G: Neurovascular factors in woundhealing in the foot skin of type 2 diabetic subjects.Diabetes Care 2007; 30: 3058.

18. Bhatwadekar AD, Guerin EP, Jarajapu YP,Caballero S, Sheridan C, Kent D, et al.: Transient

inhibition of transforming growth factor-beta1in human diabetic CD34 + cells enhances vascularreparative functions. Diabetes 2010; 59: 2010.

19. Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL,Chen J, et al.: Silk-based biomaterials. Bioma-terials 2003; 24: 401.

20. Brem H, Stojadinovic O, Diegelmann RF, Entero H,Lee B, Pastar I, et al.: Molecular markers in pa-tients with chronic wounds to guide surgical de-bridement. Mol Med 2007; 13: 30.

21. Stringer SE: The role of heparan sulphate pro-teoglycans in angiogenesis. Biochem Soc Trans2006; 34(Pt 3): 451.

22. Dalton SJ, Whiting CV, Bailey JR, Mitchell DC, andTarlton JF: Mechanisms of chronic skin ulcerationlinking lactate, transforming growth factor-beta,vascular endothelial growth factor, collagen re-modeling, collagen stability, and defective angio-genesis. J Invest Dermatol 2007; 127: 958.

23. Lauer G, Sollberg S, Cole M, Flamme I, Sturze-becher J, Mann K, et al.: Expression and prote-olysis of vascular endothelial growth factor isincreased in chronic wounds. J Invest Dermatol2000; 115: 12.

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