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1.
Sci Transl Med ; 16(752): eadf0555, 2024 Jun 19.
Article in English | MEDLINE | ID: mdl-38896604

ABSTRACT

Despite decades of effort aimed at developing clinically effective cell therapies, including mixed population mononuclear cells, to revascularize the ischemic limb, there remains a paucity of patient-based studies that inform the function and fate of candidate cell types. In this study, we showed that circulating proangiogenic/arteriogenic monocytes (PAMs) expressing the FcγIIIA receptor CD16 were elevated in patients with chronic limb-threatening ischemia (CLTI), and these amounts decreased after revascularization. Unlike CD16-negative monocytes, PAMs showed large vessel remodeling properties in vitro when cultured with endothelial cells and smooth muscle cells and promoted salvage of the ischemic limb in vivo in a mouse model of hindlimb ischemia. PAMs showed a propensity to migrate toward and bind to ischemic muscle and to secrete angiogenic/arteriogenic factors, vascular endothelial growth factor A (VEGF-A) and heparin-binding epidermal growth factor. We instigated a first-in-human single-arm cohort study in which autologous PAMs were injected into the ischemic limbs of five patients with CLTI. Greater than 25% of injected cells were retained in the leg for at least 72 hours, of which greater than 80% were viable, with evidence of enhanced large vessel remodeling in the injected muscle area. In summary, we identified up-regulation of a circulatory PAM subpopulation as an endogenous response to limb ischemia in CLTI and tested a potentially clinically relevant therapeutic strategy.


Subject(s)
Hindlimb , Ischemia , Monocytes , Neovascularization, Physiologic , Humans , Monocytes/metabolism , Animals , Ischemia/pathology , Ischemia/metabolism , Ischemia/therapy , Hindlimb/blood supply , Receptors, IgG/metabolism , Mice , Male , Vascular Endothelial Growth Factor A/metabolism , Female , Aged , Middle Aged , Cell Movement , Heparin-binding EGF-like Growth Factor/metabolism
2.
JCI Insight ; 8(24)2023 Dec 22.
Article in English | MEDLINE | ID: mdl-37847559

ABSTRACT

Use of autologous cells isolated from elderly patients with multiple comorbidities may account for the modest efficacy of cell therapy in patients with chronic limb threatening ischemia (CLTI). We aimed to determine whether proarteriogenic monocyte/macrophages (Mo/MΦs) from patients with CLTI were functionally impaired and to demonstrate the mechanisms related to any impairment. Proarteriogenic Mo/MΦs isolated from patients with CLTI were found to have an impaired capacity to promote neovascularization in vitro and in vivo compared with those isolated from healthy controls. This was associated with increased expression of human HIV-1 TAT interactive protein-2 (HTATIP2), a transcription factor known to suppress angiogenesis/arteriogenesis. Silencing HTATIP2 restored the functional capacity of CLTI Mo/MΦs, which was associated with increased expression of arteriogenic regulators Neuropilin-1 and Angiopoietin-1, and their ability to enhance angiogenic (endothelial tubule formation) and arteriogenic (smooth muscle proliferation) processes in vitro. In support of the translational relevance of our findings, silencing HTATIP2 in proarteriogenic Mo/MΦs isolated from patients with CLTI rescued their capacity to enhance limb perfusion in the ischemic hindlimb by effecting greater angiogenesis and arteriogenesis. Ex vivo modulation of HTATIP2 may offer a strategy for rescuing the functional impairment of pro-angio/arteriogenic Mo/MΦs prior to autologous delivery and increase the likelihood of clinical efficacy.


Subject(s)
Monocytes , Neovascularization, Physiologic , Animals , Mice , Humans , Aged , Monocytes/metabolism , Collateral Circulation , Muscle, Skeletal/metabolism , Mice, Knockout , Ischemia/metabolism , Transcription Factors , Acetyltransferases
3.
NPJ Regen Med ; 4: 6, 2019.
Article in English | MEDLINE | ID: mdl-30911410

ABSTRACT

Cell therapies to treat critical limb ischaemia have demonstrated only modest results in clinical trials, and this has been partly attributed to poor cell retention following their delivery directly into the ischaemic limb. The aim of this study was to determine whether alginate encapsulation of therapeutic pro-angio/arteriogenic macrophages enhances their retention and ultimately improves limb perfusion. A reproducible GMP-compliant method for generating 300 µm alginate capsules was developed to encapsulate pro-angio/arteriogenic macrophages. Longitudinal analysis revealed no detrimental effect of encapsulation on cell number or viability in vitro, and macrophages retained their pro-angio/arteriogenic phenotype. Intramuscular delivery of encapsulated macrophages into the murine ischaemic hindlimb demonstrated increased cell retention compared with injection of naked cells (P = 0.0001), and that this was associated both enhanced angiogenesis (P = 0.02) and arteriogenesis (P = 0.03), and an overall improvement in limb perfusion (P = 0.0001). Alginate encapsulation of pro-angio/arteriogenic macrophages enhances cell retention and subsequent limb reperfusion in vivo. Encapsulation may therefore represent a means of improving the efficacy of cell-based therapies currently under investigation for the treatment of limb ischaemia.

5.
Circulation ; 136(25): 2406-2416, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29054934

ABSTRACT

BACKGROUND: Radiation exposure during fluoroscopically guided interventions such as endovascular aortic repair (EVAR) is a growing concern for operators. This study aimed to measure DNA damage/repair markers in operators perfoming EVAR. METHODS: Expression of the DNA damage/repair marker, γ-H2AX and DNA damage response marker, phosphorylated ataxia telangiectasia mutated (pATM), were quantified in circulating lymphocytes in operators during the peri-operative period of endovascular (infrarenal, branched, and fenestrated) and open aortic repair using flow cytometry. These markers were separately measured in the same operators but this time wearing leg lead shielding in addition to upper body protection and compared with those operating with unprotected legs. Susceptibility to radiation damage was determined by irradiating operators' blood in vitro. RESULTS: γ-H2AX and pATM levels increased significantly in operators immediately after branched endovascular aortic repair/fenestrated endovascular aortic repair (P<0.0003 for both). Only pATM levels increased after infrarenal endovascular aortic repair (P<0.04). Expression of both markers fell to baseline in operators after 24 hours (P<0.003 for both). There was no change in γ-H2AX or pATM expression after open repair. Leg protection abrogated γ-H2AX and pATM response after branched endovascular aortic repair/fenestrated endovascular aortic repair. The expression of γ-H2AX varied significantly when operators' blood was exposed to the same radiation dose in vitro (P<0.0001). CONCLUSIONS: This is the first study to detect an acute DNA damage response in operators performing fluoroscopically guided aortic procedures and highlights the protective effect of leg shielding. Defining the relationship between this response and cancer risk may better inform safe levels of chronic low-dose radiation exposure.


Subject(s)
Aortic Aneurysm, Abdominal/surgery , DNA Damage/radiation effects , Occupational Exposure , Radiation, Ionizing , Adult , Ataxia Telangiectasia Mutated Proteins/metabolism , Endovascular Procedures , Female , Fluoroscopy , Histones/metabolism , Humans , Immunohistochemistry , Leg/radiation effects , Male , Middle Aged , Phosphorylation , Radiation Protection/instrumentation , T-Lymphocytes/metabolism , T-Lymphocytes/radiation effects
6.
J Exp Med ; 214(8): 2437-2452, 2017 Aug 07.
Article in English | MEDLINE | ID: mdl-28724617

ABSTRACT

Venous valves (VVs) prevent venous hypertension and ulceration. We report that FOXC2 and GJC2 mutations are associated with reduced VV number and length. In mice, early VV formation is marked by elongation and reorientation ("organization") of Prox1hi endothelial cells by postnatal day 0. The expression of the transcription factors Foxc2 and Nfatc1 and the gap junction proteins Gjc2, Gja1, and Gja4 were temporospatially regulated during this process. Foxc2 and Nfatc1 were coexpressed at P0, and combined Foxc2 deletion with calcineurin-Nfat inhibition disrupted early Prox1hi endothelial organization, suggesting cooperative Foxc2-Nfatc1 patterning of these events. Genetic deletion of Gjc2, Gja4, or Gja1 also disrupted early VV Prox1hi endothelial organization at postnatal day 0, and this likely underlies the VV defects seen in patients with GJC2 mutations. Knockout of Gja4 or Gjc2 resulted in reduced proliferation of Prox1hi valve-forming cells. At later stages of blood flow, Foxc2 and calcineurin-Nfat signaling are each required for growth of the valve leaflets, whereas Foxc2 is not required for VV maintenance.


Subject(s)
Connexins/genetics , Forkhead Transcription Factors/genetics , Heart Valve Diseases/etiology , Heart Valve Diseases/genetics , Mutation/genetics , Venous Valves/metabolism , Animals , Cell Proliferation/genetics , Endothelial Cells/metabolism , Gap Junctions/genetics , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Regional Blood Flow/genetics , Signal Transduction/genetics
7.
Sci Rep ; 7(1): 1501, 2017 05 04.
Article in English | MEDLINE | ID: mdl-28473691

ABSTRACT

The nucleus pulposus (NP) of the intervertebral disc (IVD) demonstrates substantial changes in cell and matrix composition with both ageing and degeneration. While recent transcriptomic profiling studies have helped define human NP cell phenotype, it remains unclear how expression of these markers is influenced by ageing or degeneration. Furthermore, cells of the NP are thought to derive from the notochord, although adult NP lacks identifiable notochordal (NC) cells. This study aimed to confirm expression of previously identified NP and NC marker genes in adult human NP cells from a range of ages and degenerate states. Importantly, using gene expression analysis (N = 60) and immunohistochemistry (N = 56) the study demonstrates expression of NP markers FoxF1, Pax-1, keratin-8/18, carbonic anhydrase-12, and NC markers brachyury, galectin-3 and CD24 in cells of the NP irrespective of age or degeneration. Our immunohistochemical data, combined with flow cytometry (N = 5) which identified a small number of CA12+Gal3+T+CD24+ cells, suggests the possible presence of a sub-population of cells with an NC-like phenotype in adult NP tissue. These findings suggest that the NP contains a heterogeneous population of cells, which may possess varied phenotypic and functional profiles and thus warrant further investigation to improve our understanding of IVD homeostasis and repair.


Subject(s)
Aging/metabolism , Biomarkers/metabolism , Intervertebral Disc Degeneration/metabolism , Intervertebral Disc Degeneration/pathology , Notochord/metabolism , Nucleus Pulposus/metabolism , Adolescent , Adult , Humans , Intervertebral Disc/metabolism , Intervertebral Disc/pathology , Middle Aged , Young Adult
8.
J Am Coll Cardiol ; 67(4): 420-431, 2016 Feb 02.
Article in English | MEDLINE | ID: mdl-26821631

ABSTRACT

BACKGROUND: Use of blood oxygenation level-dependent cardiovascular magnetic resonance (BOLD-CMR) to assess perfusion in the lower limb has been hampered by poor reproducibility and a failure to reliably detect post-revascularization improvements in patients with critical limb ischemia (CLI). OBJECTIVES: This study sought to develop BOLD-CMR as an objective, reliable clinical tool for measuring calf muscle perfusion in patients with CLI. METHODS: The calf was imaged at 3-T in young healthy control subjects (n = 12), age-matched control subjects (n = 10), and patients with CLI (n = 34). Signal intensity time curves were generated for each muscle group and curve parameters, including signal reduction during ischemia (SRi) and gradient during reactive hyperemia (Grad). BOLD-CMR was used to assess changes in perfusion following revascularization in 12 CLI patients. Muscle biopsies (n = 28), obtained at the level of BOLD-CMR measurement and from healthy proximal muscle of patients undergoing lower limb amputation (n = 3), were analyzed for capillary-fiber ratio. RESULTS: There was good interuser and interscan reproducibility for Grad and SRi (all p < 0.0001). The ischemic limb had lower Grad and SRi compared with the contralateral asymptomatic limb, age-matched control subjects, and young control subjects (p < 0.001 for all comparisons). Successful revascularization resulted in improvement in Grad (p < 0.0001) and SRi (p < 0.0005). There was a significant correlation between capillary-fiber ratio (p < 0.01) in muscle biopsies from amputated limbs and Grad measured pre-operatively at the corresponding level. CONCLUSIONS: BOLD-CMR showed promise as a reliable tool for assessing perfusion in the lower limb musculature and merits further investigation in a clinical trial.


Subject(s)
Angioplasty/methods , Ischemia/blood , Leg/blood supply , Magnetic Resonance Imaging/methods , Oxygen/blood , Adult , Aged , Aged, 80 and over , Biopsy , Disease Progression , Female , Follow-Up Studies , Humans , Ischemia/diagnosis , Ischemia/therapy , Male , Middle Aged , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Prognosis , Reproducibility of Results , Severity of Illness Index , Ultrasonography, Doppler, Duplex , Young Adult
10.
Regen Med ; 8(1): 75-87, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23259807

ABSTRACT

Low back pain is a leading cause of morbidity in developed societies and is strongly linked to degeneration of the intervertebral disc. The central nucleus pulposus (NP) region is most severely affected during disc degeneration and, consequently, is a focus for novel cell-based regenerative strategies. However, in order to develop such techniques, it is essential to first understand the biology and phenotype of the NP cells intended for repair. Microarray studies have highlighted novel NP markers that will allow a more accurate identification of cells for implantation, and along with other studies, have also revealed the potential importance of a developmental or immature NP cell phenotype in disseminating the optimal cell type for use. Additionally, the degenerative intervertebral disc is a harsh native environment and the effects of this on cells intended for implantation have yet to be fully elucidated; this is crucial for clinical translation of tissue engineered cell-based therapies.


Subject(s)
Intervertebral Disc Degeneration/pathology , Intervertebral Disc Degeneration/physiopathology , Intervertebral Disc/pathology , Intervertebral Disc/physiopathology , Regeneration , Animals , Biological Therapy , Humans , Intervertebral Disc Degeneration/therapy , Low Back Pain/therapy , Phenotype
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