Wound Healing Angiogenesis:

Innovations and Challenges in Acute

and Chronic Wound Healing
1 2 2,
Tatiana N. Demidova-Rice, Jennifer T. Durham, and Ira M. Herman *
1
Department of Radiation Oncology, Edwin L. Steele Laboratory for Tumor Biology, Massachusetts General Hospital,
Boston, MA 02114.
2
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.

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) proteolytic enzymes, (iii) activators of multiple
differentiated and progenitor cell types, and
(iv) permissible microenvironments. A central aim of wound healing research is to
‘‘convert’’ chronic, disease-impaired wounds into those that will heal.
The problem: Reduced ability to re-establish a blood supply to the injury site can Ira M. Herman

ultimately lead to wound chronicity. Submitted for publication June 27, 2011.
Basic/Clinical Science Advances: (1) Human fetal endothelial progenitor cells can *Correspondence: Department of Molecular
stimulate wound revascularization and repair following injury, as demon-strated in a novel Physiology and Pharmacology and the Center
for Innovations in Wound Healing Research,
mouse model of diabetic ischemic healing. (2) Advances in bioengineering reveal exciting Tufts University School of Medicine and
alternatives by which wound repair may be fa-cilitated via the creation of vascularized Graduate Program in Cellular and Molecular
microfluidic networks within organ constructs created ex vivo for wound implantation. (3) A Physiology, Sackler School of Graduate
Biomedical Sciences, Tufts University, Boston,
‘‘personalized’’ ap-proach to regenerative medicine may be enabled by the identification of MA 02111 (e-mail: ira.herman@tufts.edu)
protein components present within individual wound beds, both chronic and acute.

Clinical Care Relevance: Despite the development of numerous therapies, im-paired Abbreviations
angiogenesis and wound chronicity remain significant healthcare prob-lems. As such, and Acronyms
innovations in enhancing wound revascularization would lead to significant advances in EC = endothelial cell
wound healing therapeutics and patient care. EPC = endothelial
Conclusion: Insights into endothelial progenitor cell biology together with de-velopments progenitor cell
in the field of tissue engineering and molecular diagnostics should not only further advance FPC = fetal progenitor cells
our understanding of the molecular mechanisms regulating wound repair but also offer
FPC133 + = CD133-positive
innovative solutions to promote the healing of chronic and acute wounds in vivo.
fetal progenitor cells
VEGF = vascular
endothelial growth factor
BACKGROUND endothelial growth factor (VEGF). In
Angiogenesis, the formation of new turn, ECs degrade their surrounding
blood vessels from the preexist-ing extracellular matrix, begin migra-tion and
vasculature, is indispensable for cell division (proliferation), and then
successful wound healing. Post-injury, reestablish cell–cell con-tacts, forming
1
microvascular endothelial cells (ECs) new capillaries. An-other critical
that line the inner surface of blood process required for wound
vessels are activated by low oxygen revascularization is vasculo-genesis,
tension/hypoxia and proan-giogenic which relies on the mobili-zation of
factors, including vascular endothelial progenitor cells

ADVANCES IN WOUND CARE, VOLUME 1, NUMBER 1

Copyright ª 2012 by Mary Ann Liebert, Inc. DOI: 10.1089/wound.2011.0308 j 17
18 DEMIDOVA-RICE ET AL.

(EPCs) from the bone marrow to areas of regenerat-ing technologically or biologically hampered.9 Thus, the
or healing tissues. EPCs (both embryonic and adult) challenge persists to create innovative and effective
possess several stem-cell like surface markers, including therapeutics for acute and chronic wound repair.
the cell surface glycoprotein CD133, and can
2
differentiate into several cell types. Although stem cell BASIC SCIENCE CONTEXT
applications have proven beneficial in car-diovascular
A growing body of evidence has advanced our
settings,3 EPCs’ efficacy in fostering diabetic wound understanding of the mechanisms leading to non-healing,
healing remains problematic, be-cause diabetic patients chronic wounds.10 These include, but are not limited to,
4,5
possess both diminished and dysfunctional EPCs as reduced bioavailability of growth factors and receptors,
well as unfavorable wound microenvironments. As a abnormal production/ modification of matrix proteins,
need for molecular and cellular therapeutics remains diminished prolifer-ative capacity of resident cells, and
evident, alternative pathways are being explored, insufficient or impaired wound perfusion (Fig. 1). The
including the devel-opment of implantable bioengineered latter stems from EC dysfunction as well as impaired re-
microvascular networks that would enhance wound bed 11
cruitment of EPCs to the injury site. One method to
perfusion.6 Ultimately, understanding patient-specific circumvent this issue is the application of exogenous
7
meta-bolic and molecular profiles should provide potent adult or fetal progenitor cells (FPCs) to chronic wound
diagnostic tools, advancing the field toward a per- beds.12 Commonly expressing the EPC sur-face markers
sonalized regenerative medicine-based approach. CD133, CD34, and VEGF receptor-2 (KDR), these cells
can differentiate into both vascular endothelial and
2
perivascular (mural) cells. In addi-tion, CD133-positive
TARGET ARTICLES cells generate proangiogenic
1. Barcelos LS, Duplaa C, Krankel N, Graiani G,
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 diabetic ischemic ulcers by
paracrine stimulation of angiogenesis 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, and Herman IM:
Tissue equivalents based on cell-seeded biodegradable
microfluidic constructs. Materials 2010; 3: 1833.

3. Eming SA, Koch M, Krieger A, Brachvogel B,

Kreft S, Bruckner-Tuderman L, Krieg T, Shannon JD,
and Fox JW: Differential proteomic analysis
distinguishes tissue repair biomarker signatures in
wound exudates obtained from normal healing and
chronic wounds. J Proteome Res 2010; 9: 4758.

CLINICAL PROBLEM ADDRESSED

Cells and tissues are inherently dependent on the FIG. 1. Chronic wounds are often characterized by hyperglycemia, per-sistent
vasculature to supply critical nutrients and oxygen in inflammation, and growth factor and cytokine receptor deficiencies, which lead
to impaired progenitor cell recruitment and angiogenesis and delayed
exchange for metabolites. Impaired wound revas-
epithelialization. Despite the progress achieved in the development of wound
cularization can impede healing, just as insufficient healing therapeutics, significant problems persist. Novel wound healing
perfusion is a hallmark of chronic wounds. Several approaches that aim to include adult or embryonic progenitor cells or
approaches including delivery of proangiogenic growth vascularized skin grafts seem promising, yet these methods will require
factors8 have been employed to enhance wound healing; further preclinical and clinical evaluation before normalization of the chronic
wound microenvironment fosters the restoration of wound healing.
however, current treatments remain
 ANGIOGENESIS OF WOUND HEALING 19
Creation of prevascularized microfluidic
networks in biodegradable scaffolds
2
Microvascular networks with dimensions and features
soluble mediators, such as VEGF, and are TAKE-HOME MESSAGE
able to promote revascularization follow-ing Basic science advances
pathologic injury, including ischemic heart
13 Human embryo–derived CD133 + progenitor cells promote both angio-
disease. Although fetal or adult endothelial
genesis and wound healing in diabetic ischemic ulcers in mice.
progenitor cells may poten-tially offer benefits
for chronic wound suf-ferers, further study The wound healing effects of live human embryo–derived
will be needed to ensure safety and efficacy. CD133 + progenitor cells can be replicated in the absence of
Also, alternative strategies for wound cells by the use of cell-free media containing soluble factors,
revascularization are being investigated such as VEGF produced by CD133 + cells (conditioned media).
including the use of bioengineered Angiogenic stimulation is achieved via soluble factor–mediated
vascularized scaffolds.6 Linked to this, increase in endothelial survival, proliferation, and migration.
successful wound revascu-larization will Nonimmunogenic silk-based channels can support survival
require a permissive micro-environment, and growth of microvascular ECs.
which could be adjusted in a patient-specific
manner based on wound conditions The wound exudates derived from acute and chronic wounds
have dif-ferent protein composition.
determined using modern pro-teomics or
7 The molecules that were exclusively found in healing wounds
point-of-care diagnostics.
include several 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
chronic wounds 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 may provide useful tools for the personalized medicine.
resembling the human dermal microvas-culature are
being constructed using biocompatible biomaterials. 6
EXPERIMENTAL MODEL
OR MATERIAL—ADVANTAGES Such silk scaffolds can be seeded with micro- or
macrovascular-derived ECs and perfused ex vivo.
AND LIMITATIONS
Moreover, these cellularized mi-crofluidic networks can
Treatment of ischemic wounds in be layered using other dermal compartment cells or
mice using human FPCs overlaid with epithe-lial cellular components. Clearly,
Isolating human fetal aortic progenitor cells further work will
represents a considerable challenge,
12,14
technically and ethically, requiring
rigorous cell selection, recovery, and prop-
agation. For example, Barcelos and co-
authors2,12 used EC-specific anti-CD31 to
eliminate differentiated ECs and anti-CD133
antibody to enrich for progenitor
cells. To evaluate the proangiogenic potential of these
cells, a newly established mouse model of is-chemic
wound healing was used, which relies upon
pharmacologic induction of diabetes, followed by is-
chemic insult. This model recapitulates both the
hyperglycemia accompanying diabetes as well as the
ischemia-reperfusion issues characteristic of chronic
wounds. Therefore, this is an attractive model to test a
variety of cellular and molecular-based healing
modalities, including progenitor cells, under a more
clinically relevant setting.
be necessary to ensure optimal host engraftment in vivo
of such vascularized ‘‘organ equivalents.’’

Analysis of protein profiles of acute

and chronic wounds
Wound exudates were noninvasively collected from
either acute or chronic (venous) wounds.7 Re-
presentative samples were subjected to protein
identification 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 intensive effort of data analysis as
hundreds of proteins are identified in this manner, and a
likely omission of potentially important yet
underrepresented targets undetectable using mass
spectrometry techniques.

DISCUSSION OF FINDINGS
AND RELEVANT LITERATURE
During normal wound healing, EPCs are effec-tively
recruited to the remodeling microcircula-tion, thus
leading to wound revascularization
20 DEMIDOVA-RICE ET AL.

and timely healing. This response is likely to be cell applications for wound healing. As an alter-native,
dampened in diabetic ulceration. Indeed, it has been the delivery of biomaterial scaffolds to fos-ter host-
recently demonstrated that EPCs from nor-mal but not specific recovery has been suggested. For example, silk
diabetic patients contribute to postis-chemic 6
fibroin, prepared as previously de-scribed, is routinely
4
revascularization. Diabetic EPCs are both lower in used for adult progenitor cell growth and
number5 and dysfunctional, displaying a shift toward a differentiation.19 Moreover, such bio-materials can be
15 fabricated into complex designs that mimic vascular
proinflammatory phenotype. Nor-mal adult and/or
FPCs (i) can differentiate into several cell types and (ii) branching patterns in vivo. In turn, the silk-based
have stimulatory effects on biological processes and are scaffolds can be populated with competent differentiated
therefore likely to be beneficial for chronic wound ECs, with fluid flow as a patterning guide. Described by
6
patients and those with impaired perfusion. However, Borenstein and coauthors, biodegradable microfluidic
successful uti-lization of stem cells for chronic wound constructs were seeded with human microvascular ECs,
healing still needs further development and optimization. which remained viable and retained their cell sur-face
In a recent study, fetal CD133-positive cells (FPC133 + ), markers for over a week. Additional modifi-cations
isolated from human fetal aortas and expanded as include coculturing with perivascular cells as well as
previously described,2 were used to stimulate is-chemic inclusion of a keratinocyte cell layer to create tissue
wound healing in diabetic mice using a collagen-based equivalents for implantation. Although the successful
12 implantation of such microfluidic devices into a wound
delivery system. Both FPC133 + and FPC133 +
bed in vivo has not been yet described, beneficial effects
-conditioned media accelerate rates of wound closure, on wound revascularization and healing are anticipated.
increase EC proliferation, and promote wound
revascularization. Strikingly, a minimal number of
vascular-associated FPC133 + are observed in wounds at Successful wound revascularization is largely
3 days postinjury/trans-plantation, yet the proangiogenic dependent on a permissive wound microenviron-ment.
effects persisted for 7 days, suggesting an indirect However, the identification and standardi-zation of the
(paracrine) mechanism that sustains FPC functionality molecular profiles of the individual wounds remains
post-injury. In fact, several soluble mediators, including challenging. Recently, a concept of ‘‘wound bar coding’’
VEGF, interleukin-6, interleukin-8, monocyte was proposed20 to provide a classification scheme based
chemoattractant protein, and granulocyte-colony on gene expression patterning by resident or peripheral
stimulating factor, have been validated as produced by wound bed cells. The levels of gene activity can be
FPC133 + .12 This observation provides an im-portant assessed using modern RNA microarray technologies
insight into the mechanisms of stem cell– mediated and presented in a ‘‘heat map’’ style, depicting both up-
repair. Of note, CD133 + cells are found in several and downregulated genes. This strategy provides potent
16
human cancers ; therefore, their delivery to wounds information, which may both guide wound debridement
might be undesirable. On the other hand, proangiogenic and lead to appropriate selection of treatment strategies
cytokines generated by CD133 + cells provides a means based on individual patient needs (personalized
to replace live progenitor cells with their conditioned medicine). However, several potential drawbacks include
media. Although cell-free preparations containing the requirement of multiple biopsies from numerous
bioactive molecules demon-strate wound healing locations and the inability to assay for specific proteins
within a wound. Therefore, a more conducive option
potential in animal models,12 a major obstacle to this
might be the noninvasive probing of the wound environ-
therapy is the insufficient growth factor receptor density ment from wound exudates as was reported by Eming
and responsiveness of cells residing within the wound and coauthors.7 This test not only enables repetitive
bed, as is often the case among chronic wound monitoring of gene expression patterns but also
17
sufferers. Alternative strategies include modification of determines protein composition of wound
patient-specific EPCs, themselves, through inhibition of microenvironments and thus might provide pow-erful
certain cell signaling pathways to increase reparative predictions of the actual conditions within the wound. A
func-tion.18 All together, progenitor cell therapy and/or recent study of comparative proteomics of acute and
appropriate modifications could prove beneficial to chronic wounds using this approach 7 has demonstrated
promote a chronic healing phenotype to an acute re- that the nonhealing ulcers have de-creased amount of
sponse. Additional work is needed to delineate cell type, several extracellular matrix components, namely, heparan
modification protocol, and mode of delivery. sulfate proteoglycan and collagen type I, both of which are
critical for

Currently, no consensus exists regarding the safety

and efficacy of exogenous/endogenous stem
 ANGIOGENESIS OF WOUND HEALING 21

21,22
normal wound revascularization. Moreover, this tious disease transmission, exist. Additional re-search
study confirms a previously established and significant remains to clarify progenitor cell biological processes
increase in production of multiple prote-ases, which are and prevent undesirable effects of cell-based therapies.
detrimental for both wound extra-cellular matrix
components and proangiogenic growth factors. 23 In Progenitor cells can be differentiated in vitro and
addition, it identifies species that are exclusively present delivered into the wounds within bioma-terial-based
in acute healing, such as the serine protease thrombin and scaffolds, thus reducing the risk of undesirable cell
the antimicrobial dermicidin. Finally, these authors differentiation in vivo.
report the identi-fication of certain proteins, which have
Combination of RNA- and protein-based di-
never been previously associated with wound healing,
agnostic tools could be used to direct suc-cessful
including the matrix molecule olfactomedin-4. Together,
wound revascularization and healing.
these results shed light on how the normalization of
wound microenvironment in a patient-specific manner,
which in turn could be directed by microarray and
20 FUTURE DEVELOPMENT OF INTEREST
proteomic analysis,7 would culminate in the promo-tion The use of adult and/or embryonic endothelial
of wound revascularization or healing. progenitor cells in combination with biocompatible
artificial microvascular networks could provide a useful
tool for revascularization and enhancement of wound
healing. Optimized protocols designed to ob-tain and
INNOVATION expand patient-compatible stem cells should be devised
In recent decades, embryonic and adult endo-thelial and would continue to add to the regimen of personalized
progenitor cells have been proposed to have therapeutic medicine. Additional studies using novel animal models
potential. However, it remains to be shown whether these will determine the cellular and molecular mechanisms
cells are applicable for treatment of ischemic diabetic that underlie acute and chronic healing and provide a
wounds. The work of Barcelos and coauthors12 better understanding of the basic biology underlying
demonstrates that, in fact, FPC133 + have proangiogenic stem cell application, which would demonstrate both the
properties in this model and thus might be a useful tool safety and effi-cacy of this treatment for the clinic.
for treatment of ischemic wounds in diabetic patients. Insightful crea-tion of therapeutic interventions as well
Further, endothelialized microvascular networks based as powerful predictive tools would positively affect the
6
on degradable silk scaffolds may provide a valuable outcomes for managing and healing chronic wounds.
platform for future development of vas-cularized Ulti-mately, these approaches should be based on per-
biomaterials. Finally, identification of protein sonalized approaches to wound diagnosis and innovative
components within wound microenviron-ments advances treatment modalities in efforts to decrease the chronic
the field reach of the development of personalized wound burden of the population.
approaches to wound care.

ACKNOWLEDGEMENTS AND FUNDING

SOURCES
CAUTION, CLINICAL REMARKS,
This work was funded by NIH grants (EY15125 and
AND RECOMMENDATIONS
EY19533).
Although undifferentiated human-derived FPC133 +
could be considered a helpful tool for chronic wound
revascularization, significant risks to the patients, AUTHOR DISCLOSURE AND GHOSTWRITING
including risk of cancer development and infec- I.M. Herman is a consultant for Healthpoint, Inc.

REFERENCES
1. Singer AJ and Clark RA: Cutaneous wound heal- 3. Asahara T, Murohara T, Sullivan A, Silver 5. Brunner S, Hoellerl F, Schmid-Kubista KE, Zeiler
ing. N Engl J Med 1999; 341: 738. M, van der Zee R, Li T, et al.: Isolation of F, Schernthaner G, Binder S, et al.: Circulating an-
putative pro-genitor endothelial cells for giopoietic cells and diabetic retinopathy in T2DM
2. Invernici G, Emanueli C, Madeddu P, Cristini S,
angiogenesis. Science 1997; 275: 964. patients with and without macrovascular disease.
Gadau S, Benetti A, et al.: Human fetal aorta
Invest Ophthalmol Vis Sci 2011; 52: 4655.
contains vascular progenitor cells 4. Caballero S, Sengupta N, Afzal A, Chang KH, Li
capable of inducing vasculogenesis, Calzi S, Guberski DL, et al.: Ischemic vascular 6. Borenstein JT, Megley K, Wall K, Pritchard EM,
angiogenesis, and myogenesis in vitro damage can be repaired by healthy, but not dia- Truong D, Kaplan DL, et al.: Tissue equivalents
and in a murine model of peripheral betic, endothelial progenitor cells. Diabetes 2007; based on cell-seeded biodegradable microfluidic
ischemia. Am J Pathol 2007; 170: 1879. 56:960. constructs. Materials 2010; 3: 1833.
22 DEMIDOVA-RICE ET AL.

7. Eming SA, Koch M, Krieger A, Brachvogel B, Kreft 13. Adler DS, Lazarus H, Nair R, Goldberg JL, Greco inhibition of transforming growth factor-beta1 in
S, Bruckner-Tuderman L, et al.: Differential pro- NJ, Lassar T, et al.: Safety and efficacy of bone human diabetic CD34 + cells enhances vascular
teomic analysis distinguishes tissue repair bio- marrow-derived autologous CD133 + stem cell reparative functions. Diabetes 2010; 59: 2010.
marker signatures in wound exudates obtained therapy. Front Biosci (Elite Ed) 2011; 3: 506.
from normal healing and chronic wounds. J Pro-
19. Altman GH, Diaz F, Jakuba C, Calabro T,
14. Gucciardo L, Lories R, Ochsenbein-Kolble N, Horan RL, Chen J, et al.: Silk-based
teome Res 2010; 9: 4758.
Done’ E, Zwijsen A, and Deprest J: Fetal biomaterials. Bioma-terials 2003; 24: 401.
8. Leahy PJ and Lawrence WT: Biologic enhance- mesenchymal stem cells: isolation, properties
ment of wound healing. Clin Plast Surg 2007; and potential use in perinatology and 20. Brem H, Stojadinovic O, Diegelmann RF, Entero
H, Lee B, Pastar I, et al.: Molecular markers in pa-
34:659. regenerative medicine. BJOG 2009; 116: 166.
tients with chronic wounds to guide surgical de-
9. Schultz GS, Davidson JM, Kirsner RS, Bornstein P, and 15. Loomans CJ, van Haperen R, Duijs JM, Verseyden C, de bridement. Mol Med 2007; 13: 30.
Herman IM: Dynamic reciprocity in the wound Crom R, Leenen PJ, et al.: Differentiation of bone marrow-
microenvironment. Wound Repair Regen 2011; derived endothelial progenitor cells is shifted into a 21. Stringer SE: The role of heparan
sulphate pro-teoglycans in angiogenesis.
19:134. proinflammatory phenotype by hyperglycemia. Mol Med
2009; 15: 152.
Biochem Soc Trans 2006; 34(Pt 3): 451.
10. Falanga V: Wound healing and its impairment in
16. Monzani E, Facchetti F, Galmozzi E, Corsini E, Benetti A, 22. Dalton SJ, Whiting CV, Bailey JR, Mitchell DC, and Tarlton
the diabetic foot. Lancet 2005; 366: 1736.
Cavazzin C, et al.: Melanoma contains CD133 and ABCG2 JF: Mechanisms of chronic skin ulceration linking lactate,
11. Liu ZJ and Velazquez OC: Hyperoxia, endothelial positive cells with enhanced tumourigenic potential. Eur J transforming growth factor-beta, vascular endothelial
progenitor cell mobilization, and diabetic wound Cancer 2007; 43: 935. growth factor, collagen re-modeling, collagen stability, and
healing. Antioxid Redox Signal 2008; 10: 1869. defective angio-genesis. J Invest Dermatol 2007; 127: 958.
17. Krishnan ST, Quattrini C, Jeziorska M, Malik RA,
12. Barcelos LS, Duplaa C, Krankel N, Graiani G, and Rayman G: Neurovascular factors in wound
In-vernici G, Katare R, et al.: Human CD133 + healing in the foot skin of type 2 diabetic subjects. 23. Lauer G, Sollberg S, Cole M, Flamme I,
pro-genitor cells promote the healing of Diabetes Care 2007; 30: 3058. Sturze-becher J, Mann K, et al.: Expression
diabetic ischemic ulcers by paracrine and prote-olysis of vascular endothelial
stimulation of an-giogenesis and activation of 18. Bhatwadekar AD, Guerin EP, Jarajapu YP,
growth factor is increased in chronic
wnt signaling. Circ Res 2009; 104: 1095. Caballero S, Sheridan C, Kent D, et al.: Transient wounds. J Invest Dermatol 2000; 115: 12.