Revascularization remains a challenge for regenerative medicine strategies. Extensive research has been done to identify key moments of the dynamic wound healing cascade where targeted therapies can elicit a proregenerative response. However, the influence of oxygenation, temperature, and their temporal variation during healing are often challenging to promote tissue regeneration. This study investigated the effects of temporally varied oxygenation and temperature conditions on angiogenesis using an in vitro model of rat-derived, intact microvascular fragments in a collagen type-I hydrogel. By generating culture conditions that are similar to the accepted wound healing time course, the angiogenic response depended critically on both the timing of stimulus initiation and the magnitude of deviation from model conditions. Dynamic stimuli activated distinct biological pathways, as evidenced by qPCR analysis, revealing mechanistic links between environmental perturbations and the angiogenic response. This work emphasizes the need for regenerative medicine strategies to consider varying environmental stimuli to improve revascularization outcomes.
Impact Statement
This work demonstrated the impact of time-varying oxygenation and temperature conditions on self-assembling three-dimensional microvascular networks in vitro that mimic the physiological time course of wound healing. These findings suggest an important temporal relationship in angiogenesis where unresolved oxygen and temperature environments inhibit vascular network formation, cellular viability, proliferation, and environment-specific transcriptional factors.
KrishnanL, ChangCC, NunesSS, et al.Manipulating the microvasculature and its microenvironment. Crit Rev Biomed Eng, 2013; 41(2):91–123; doi: 10.1615/critrevbiomedeng.2013008077
3.
GonzalezACO, CostaTF, AndradeZA, et al.Wound healing—a literature review. An Bras Dermatol, 2016; 91(5):614–620; doi: 10.1590/abd1806-4841.20164741
4.
VelnarT, BaileyT, SmrkoljV. The wound healing process: An overview of the cellular and molecular mechanisms. J Int Med Res, 2009; 37(5):1528–1542; doi: 10.1177/147323000903700531
5.
SorgH, TilkornDJ, MirastschijskiU, et al.Panta rhei: Neovascularization, angiogenesis and nutritive perfusion in wound healing. Eur Surg Res, 2018; 59(3–4):232–241; doi: 10.1159/000492410
6.
VelazquezOC. Angiogenesis & vasculogenesis: Inducing the growth of new blood vessels and wound healing by stimulation of bone marrow derived progenitor cell mobilization and homing. J Vasc Surg, 2007; 45(Suppl A):A39–A47; doi: 10.1016/j.jvs.2007.02.068
7.
BoerckelJD, UhrigBA, WillettNJ, et al.Mechanical regulation of vascular growth and tissue regeneration in vivo. Proc Natl Acad Sci U S A, 2011; 108(37):E674–E680; doi: 10.1073/pnas.1107019108
8.
BauerSM, BauerRJ, VelazquezOC. Angiogenesis, vasculogenesis, and induction of healing in chronic wounds. Vasc Endovascular Surg, 2005; 39(4):293–306; doi: 10.1177/153857440503900401
KrishnanL, HoyingJB, NguyenH, et al.Interaction of angiogenic microvessels with the extracellular matrix. Am J Physiol Heart Circ Physiol, 2007; 293(6):H3650–H3658; doi: 10.1152/ajpheart.00772.2007
11.
PittmanRN. Oxygen gradients in the microcirculation. Acta Physiol (Oxf), 2011; 202(3):311–322; doi: 10.1111/j.1748-1716.2010.02232.x
12.
PhanTX, SahibzadaN, AhernGP. Arteries are finely tuned thermosensors regulating myogenic tone and blood flow. bioRxiv, 2023:2023.03.22.532099; doi: 10.1101/2023.03.22.532099
13.
SenCK. Wound healing essentials: Let there be oxygen. Wound Repair Regen, 2009; 17(1):1–18; doi: 10.1111/j.1524-475X.2008.00436.x
14.
YoussefK, UllahA, RezaiP, et al.Recent advances in biosensors for real time monitoring of pH, temperature, and oxygen in chronic wounds. Mater Today Bio, 2023; 22:100764; doi: 10.1016/j.mtbio.2023.100764
15.
DrouinG, CoutureV, LauzonM-A, et al.Muscle injury-induced hypoxia alters the proliferation and differentiation potentials of muscle resident stromal cells. Skelet Muscle, 2019; 9(1):18; doi: 10.1186/s13395-019-0202-5
16.
ScheererN, DehneN, StockmannC, et al.Myeloid hypoxia-inducible factor-1α is essential for skeletal muscle regeneration in mice. J Immunol, 2013; 191(1):407–414; doi: 10.4049/jimmunol.1103779
17.
DarbyIA, HewitsonTD. Hypoxia in tissue repair and fibrosis. Cell Tissue Res, 2016; 365(3):553–562; doi: 10.1007/s00441-016-2461-3
18.
MohyeldinA, Garzón-MuvdiT, Quiñones-HinojosaA. Oxygen in stem cell biology: A critical component of the stem cell niche. Cell Stem Cell, 2010; 7(2):150–161; doi: 10.1016/j.stem.2010.07.007
19.
MengerMM, KörbelC, BauerD, et al.Photoacoustic imaging for the study of oxygen saturation and total hemoglobin in bone healing and non-union formation. Photoacoustics, 2022; 28:100409; doi: 10.1016/j.pacs.2022.100409
20.
DerwinR, PattonD, StrappH, et al.The effect of inflammation management on pH, temperature, and bacterial burden. Int Wound J, 2023; 20(4):1118–1129; doi: 10.1111/iwj.13970
KennyGP, McGinnR. Restoration of thermoregulation after exercise. J Appl Physiol (1985), 2017; 122(4):933–944; doi: 10.1152/japplphysiol.00517.2016
23.
TikhonovaIV, TankanagAV, ChemerisNK. Age-related changes of skin blood flow during postocclusive reactive hyperemia in human. Skin Res Technol, 2013; 19(1):e174–e181; doi: 10.1111/j.1600-0846.2012.00624.x
24.
HuangJ, FanC, MaY, et al.Exploring thermal dynamics in wound healing: The impact of temperature and microenvironment. Clin Cosmet Investig Dermatol, 2024; 17:1251–1258; doi: 10.2147/CCID.S468396
25.
SawajiY, SatoT, TakeuchiA, et al.Anti-angiogenic action of hyperthermia by suppressing gene expression and production of tumour-derived vascular endothelial growth factor in vivo and in vitro. Br J Cancer, 2002; 86(10):1597–1603; doi: 10.1038/sj.bjc.6600268
26.
GoertzO, BaerreiterS, RingA, et al.Determination of microcirculatory changes and angiogenesis in a model of frostbite injury in vivo. J Surg Res, 2011; 168(1):155–161; doi: 10.1016/j.jss.2009.07.012
27.
HeG, CaoY, TangJ, et al.Assessing skin healing and angiogenesis of deep burns in vivo using two-photon microscopy in mice. Front Phys, 2022; 10; doi: 10.3389/fphy.2022.931419
28.
ShiZ, YaoC, ShuiY, et al.Research progress on the mechanism of angiogenesis in wound repair and regeneration. Front Physiol, 2023; 14:1284981; doi: 10.3389/fphys.2023.1284981
29.
HsuH-H, KoP-L, PengC-C, et al.Studying sprouting angiogenesis under combination of oxygen gradients and co-culture of fibroblasts using microfluidic cell culture model. Mater Today Bio, 2023; 21:100703; doi: 10.1016/j.mtbio.2023.100703
30.
RocaC, PrimoL, ValdembriD, et al.Hyperthermia inhibits angiogenesis by a plasminogen activator inhibitor 1-dependent mechanism. Cancer Res, 2003; 63(7):1500–1507.
31.
LaschkeMW, HeßA, ScheuerC, et al.Subnormothermic short-term cultivation improves the vascularization capacity of adipose tissue-derived microvascular fragments. J Tissue Eng Regen Med, 2019; 13(2):131–142; doi: 10.1002/term.2774
32.
KuhlenhoelterAM, KimK, NeffD, et al.Heat therapy promotes the expression of angiogenic regulators in human skeletal muscle. Am J Physiol Regul Integr Comp Physiol, 2016; 311(2):R377–R391; doi: 10.1152/ajpregu.00134.2016
33.
LemieuxP, RoudierE, BirotO. Angiostatic freeze or angiogenic move? Acute cold stress prevents angiokine secretion from murine myotubes but primes primary endothelial cells for greater migratory capacity. Front Physiol, 2022; 13:975652.
34.
MajmundarAJ, WongWJ, SimonMC. Hypoxia inducible factors and the response to hypoxic stress. Mol Cell, 2010; 40(2):294–309; doi: 10.1016/j.molcel.2010.09.022
35.
LiL, SunY, HeH, et al.Quantitative assessment of angiogenesis in skin wound healing by multi-optical imaging techniques. Front Phys, 2022; 10; doi: 10.3389/fphy.2022.894901
36.
WeickJW, KangH, LeeL, et al.Direct measurement of tissue oxygenation as a method of diagnosis of acute compartment syndrome. J Orthop Trauma, 2016; 30(11):585–591; doi: 10.1097/BOT.0000000000000651
37.
HansenEN, ManzanoG, KandemirU, et al.Comparison of tissue oxygenation and compartment pressure following tibia fracture. Injury, 2013; 44(8):1076–1080; doi: 10.1016/j.injury.2012.11.012
38.
ChapinAC, ArringtonLJ, BernardsJR, et al.Thermoregulatory and metabolic demands of naval special warfare divers during a 6-h Cold-Water training dive. Front Physiol, 2021; 12:674323; doi: 10.3389/fphys.2021.674323
39.
AbaciHE, TruittR, LuongE, et al.Adaptation to oxygen deprivation in cultures of human pluripotent stem cells, endothelial progenitor cells, and umbilical vein endothelial cells. Am J Physiol Cell Physiol, 2010; 298(6):C1527–C1537; doi: 10.1152/ajpcell.00484.2009
40.
BottensteinJE, SatoGH. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A, 1979; 76(1):514–517; doi: 10.1073/pnas.76.1.514
41.
BaumgardnerJE, OttoCM. In vitro intermittent hypoxia: Challenges for creating hypoxia in cell culture. Respir Physiol Neurobiol, 2003; 136(2–3):131–139; doi: 10.1016/S1569-9048(03)00077-6
42.
RyanS, TaylorCT, McNicholasWT. Selective activation of inflammatory pathways by intermittent hypoxia in obstructive sleep apnea syndrome. Circulation, 2005; 112(17):2660–2667; doi: 10.1161/CIRCULATIONAHA.105.556746
43.
LaBelleSA, DinkinsSS, HoyingJB, et al.Matrix anisotropy promotes angiogenesis in a density-dependent manner. Am J Physiol Heart Circ Physiol, 2022; 322(5):H806–H818; doi: 10.1152/ajpheart.00072.2022
44.
KohJ, ZhangR, RomanS, et al.Ex vivo intact tissue analysis reveals alternative calcium-sensing behaviors in parathyroid adenomas. J Clin Endocrinol Metab, 2021; 106(11):3168–3183; doi: 10.1210/clinem/dgab524
45.
SilverN, BestS, JiangJ, et al.Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol, 2006; 7:33; doi: 10.1186/1471-2199-7-33
46.
LeeS-Y, ChoeY-H, HanJ-H, et al.HPRT1 most suitable reference gene for accurate normalization of mRNA expression in canine dermal tissues with radiation therapy. Genes (Basel), 2022; 13(11):1928; doi: 10.3390/genes13111928
47.
DobkeM, PetersonDR, MatternR-H, et al.Microvascular tissue as a platform technology to modify the local microenvironment and influence the healing cascade. Regen Med, 2020; 15(2):1313–1328; doi: 10.2217/rme-2019-0139
48.
XuY, KongX, LiJ, et al.Mild Hypoxia enhances the expression of HIF and VEGF and triggers the response to injury in rat kidneys. Front Physiol, 2021; 12:690496; doi: 10.3389/fphys.2021.690496
49.
MartinezC-A, BalN, CistulliPA, et al.Intermittent hypoxia enhances the expression of HIF1A by increasing the quantity and catalytic activity of KDM4A-C and demethylating H3K9me3 at the HIF1A Locus. bioRxiv, 2021; doi: 10.1101/2021.07.25.4537262021.07.25.453726;
50.
ChangCC, NunesSS, SiboleSC, et al.Angiogenesis in a microvascular construct for transplantation depends on the method of chamber circulation. Tissue Eng Part A, 2010; 16(3):795–805; doi: 10.1089/ten.tea.2009.0370
51.
SillenM, DeclerckPJ. A narrative review on plasminogen activator Inhibitor-1 and Its (Patho)Physiological Role: To target or not to target? Int J Mol Sci, 2021; 22(5):2721; doi: 10.3390/ijms22052721
52.
AirdAL, NevittCD, ChristianK, et al.Adipose—derived stromal vascular fraction cells isolated from old animals exhibit reduced capacity to support the formation of microvascular networks. Exp Gerontol, 2015; 63:18–26; doi: 10.1016/j.exger.2015.01.044
53.
LangA, CollinsJM, NijsureMP, et al.Local erythropoiesis directs oxygen availability in bone fracture repair. bioRxiv, 2025:2025.01.10.632440; doi: 10.1101/2025.01.10.6324402025.01.10.632440;
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
