Laser Doppler Perfusion Index Research Articles

MICROCIRCULATORY ALTERATIONS IN STABLE CORONARY ARTERY DISEASE PATIENTS WITH CONCOMITANT COVID-19.

The aim: To evaluate the alterations in microcirculation of stable coronary artery disease (SCAD) patients with concomitant COVID-19. Materials and methods: The cross-sectional study analyzed the data from 80 patients, being subdivided as follows: group 1 (G1) - SCAD without COVID-19 (n=30); group 2 (G2) - SCAD with concomitant COVID-19 (n=25); group 3 (G3) - COVID-19 without SCAD (n=25). The control group included 30 relatively healthy volunteers. The state of microcirculation was assessed by nailfold videocapillaroscopy (NVC) and laser Doppler flowmetry (LDF). Results: NVC data from G2 revealed the sings of capillary bed remodeling, along with the most pronounced decrease in capillary (arteriolar part of the loop) blood flow velocity (vs. G1 and G3). LDF data from G2 were evident for the alterations in both endothelium-dependent and -independent mechanisms of microvascular flow regulation. The 72 % of G2 constituted the cases of microcirculatory hemodynamic «congestion-stasis» (MHCS) type (characterized by the decreased laser Doppler perfusion index and reduced endothelium-dependent microvascular reactivity [MVR]), and the cases of mixed type with reduced MVR. The pooled hyporeactive profile (of both MHCS type and a mixed type with reduced MVR) demonstrated the higher frequency of G2 patients (40 %), as against 11 % in the pooled alternative hemodynamic group (p<0,001) (included 80 % of cases with preserved MVR). Conclusions: G2 profile demonstrated the predomination of patients, possessing a MHCS type or a mixed type with reduced MVR. The pooled microcirculatory hyporeactive profile was presented with G2 cases to a greater extent, than in the pooled profile with predominantly preserved MVR.

Astragaloside IV promotes the angiogenic capacity of adipose-derived mesenchymal stem cells in a hindlimb ischemia model by FAK phosphorylation via CXCR2

BackgroundTherapeutic angiogenesis by transplantation of autologous/allogeneic adipose stem cells (ADSCs) is a potential method for the treatment of critical limb ischemia (CLI). However, the therapeutic efficiency is limited by poor viability, adhesion, migration and differentiation after cell transplantation into the target area. Astragaloside IV (AS-IV), one of the main active components of Astragalus, has been widely used in the treatment of ischemic diseases and can promote cell proliferation and angiogenesis. However, there is no report on the effect of AS-IV on ADSCs and its effect on hindlimb ischemia through cell transplantation. PurposeThe purpose of this study was to elucidate that AS-IV pretreatment enhances the therapeutic effect of ADSC on critical limb ischemia, and to characterize the underlying molecular mechanisms. MethodsADSCs were obtained and pretreated with the different concentration of AS-IV. In vitro, we analyzed the influence of AS-IV on ADSC proliferation, migration, angiogenesis and recruitment of human umbilical vein endothelial cells (HUVECs) and analyzed the relevant molecular mechanism. In vivo, we injected drug-pretreated ADSCs into a Matrigel or hindlimb ischemia model and evaluated the therapeutic effect by the laser Doppler perfusion index, immunofluorescence, and histopathology. ResultsIn vitro experiments showed that AS-IV improved ADSC migration, angiogenesis and endothelial recruitment. The molecular mechanism may be related to the upregulation of CXC receptor 2 (CXCR2) to promote the phosphorylation of focal adhesion kinase (FAK). In vivo, Matrigel plug assay showed that ADSCs pretreated with AS-IV have stronger angiogenic potential. The laser Doppler perfusion index of the hindlimbs of mice in the ADSC/AS-IV group was significantly higher than the laser Doppler perfusion index of the hindlimbs of mice of the ADSC group and the control group, and the microvessel density was significantly increased. ConclusionOur results demonstrate that AS-IV pretreatment of ADSC improves their therapeutic efficacy in ameliorating severe limb exclusion symptomology through CXCR2 induced FAK phosphorylation, which will bring new insights into the treatment of severe limb ischemia.

Exosomes derived from adipose-derived stem cells overexpressing glyoxalase-1 protect endothelial cells and enhance angiogenesis in type 2 diabetic mice with limb ischemia

BackgroundDiabetic limb ischemia is a clinical syndrome and refractory to therapy. Our previous study demonstrated that adipose-derived stem cells (ADSCs) overexpressing glyoxalase-1 (GLO-1) promoted the regeneration of ischemic lower limbs in diabetic mice, but low survival rate, difficulty in differentiation, and tumorigenicity of the transplanted cells restricted its application. Recent studies have found that exosomes secreted by the ADSCs have the advantages of containing parental beneficial factors and exhibiting non-immunogenic, non-tumorigenic, and strong stable characteristics.MethodsADSCs overexpressing GLO-1 (G-ADSCs) were established using lentivirus transfection, and exosomes secreted from ADSCs (G-ADSC-Exos) were isolated and characterized to coculture with human umbilical vein endothelial cells (HUVECs). Proliferation, apoptosis, migration, and tube formation of the HUVECs were detected under high-glucose conditions. The G-ADSC-Exos were injected into ischemic hindlimb muscles of type 2 diabetes mellitus (T2DM) mice, and the laser Doppler perfusion index, Masson’s staining, immunofluorescence, and immunohistochemistry assays were adopted to assess the treatment efficiency. Moreover, the underlying regulatory mechanisms of the G-ADSC-Exos on the proliferation, migration, angiogenesis, and apoptosis of the HUVECs were explored.ResultsThe G-ADSC-Exos enhanced the proliferation, migration, tube formation, and anti-apoptosis of the HUVECs in vitro under high-glucose conditions. After in vivo transplantation, the G-ADSC-Exo group showed significantly higher laser Doppler perfusion index, better muscle structural integrity, and higher microvessel’s density than the ADSC-Exo and control groups by Masson’s staining and immunofluorescence assays. The underlying mechanisms by which the G-ADSC-Exos protected endothelial cells both in vitro and in vivo might be via the activation of eNOS/AKT/ERK/P-38 signaling pathways, inhibition of AP-1/ROS/NLRP3/ASC/Caspase-1/IL-1β, as well as the increased secretion of VEGF, IGF-1, and FGF.ConclusionExosomes derived from adipose-derived stem cells overexpressing GLO-1 protected the endothelial cells and promoted the angiogenesis in type 2 diabetic mice with limb ischemia, which will be a promising clinical treatment in diabetic lower limb ischemia.

Netrin-1 improves adipose-derived stem cell proliferation, migration, and treatment effect in type 2 diabetic mice with sciatic denervation

BackgroundDiabetic peripheral neurovascular diseases (DPNVs) are complex, lacking effective treatment. Autologous/allogeneic transplantation of adipose-derived stem cells (ADSCs) is a promising strategy for DPNVs. Nonetheless, the transplanted ADSCs demonstrate unsatisfying viability, migration, adhesion, and differentiation in vivo, which reduce the treatment efficiency. Netrin-1 secreted as an axon guidance molecule and served as an angiogenic factor, demonstrating its ability in enhancing cell proliferation, migration, adhesion, and neovascularization.MethodsADSCs acquired from adipose tissue were modified by Netrin-1 gene (NTN-1) using the adenovirus method (N-ADSCs) and proliferation, migration, adhesion, and apoptosis examined under high-glucose condition. The sciatic denervated mice (db/db) with type 2 diabetes mellitus (T2DM) were transplanted with N-ADSCs and treatment efficiency assessed based on the laser Doppler perfusion index, immunofluorescence, and histopathological assay. Also, the molecular mechanisms underlying Netrin-1-mediated proliferation, migration, adhesion, differentiation, proangiogenic capacity, and apoptosis of ADSCs were explored.ResultsN-ADSCs improved the proliferation, migration, and adhesion and inhibited the apoptosis of ADSCs in vitro in the condition of high glucose. The N-ADSCs group demonstrated an elevated laser Doppler perfusion index in the ADSCs and control groups. N-ADSCs analyzed by immunofluorescence and histopathological staining demonstrated the distribution of the cells in the injected limb muscles, indicating chronic ischemia; capillaries and endothelium were formed by differentiation of N-ADSCs. The N-ADSCs group showed a significantly high density of the microvessels than the ADSCs group. The upregulation of AKT/PI3K/eNOS/P-38/NF-κB signaling pathways and secretion of multiple growth factors might explain the positive effects of Netrin-1 on ADSCs.ConclusionThe overexpression of Netrin-1 in ADSCs improves proliferation, migration, and treatment effect in type 2 diabetic mice with sciatic denervation, which directs the clinical treatment of patients with DPNVs.

Periostin enhances adipose-derived stem cell adhesion, migration, and therapeutic efficiency in Apo E deficient mice with hind limb ischemia.

IntroductionTherapeutic angiogenesis by transplantation of autologous/allogeneic adipose-derived stem cells (ADSCs) is a potential approach for severe ischemic diseases. However, poor viability, adhesion, migration and differentiation limit the therapeutic efficiency after the cells were transplanted into the targeted area. Periostin, an extracellular matrix protein, exhibits a critical role in wound repair as well as promotes cell adhesion, survival, and angiogenesis.MethodADSCs were obtained and genetically engineered with periostin gene (P-ADSCs). The viability, proliferation, migration, and apoptosis of P-ADSCs under hypoxia were analyzed. Moreover, P-ADSCs were implanted into Apo E deficient mice with hind limb ischemia. The Laser Doppler perfusion index, immunofluorescence, and histological pathology assay were tested to evaluate the therapeutic effects. The associated molecular mechanism of periostin on the proliferation, adhesion, migration, and differentiation of ADSCs was also analyzed.ResultsThe in vitro studies have shown that periostin-transfected ADSCs (P-ADSCs) promoted viability, proliferation, and migration of ADSCs. Apoptosis of ADSCs was inhibited under hypoxic conditions. The Laser Doppler perfusion index was significantly higher in the P-ADSCs group compared with that in the ADSC and control groups after 4 weeks. Immunofluorescence and histological pathology assay showed that the P-ADSCs were in and around the ischemic sites, and some cells differentiated into capillaries and endothelium. Microvessel densities were significantly improved in P-ADSCs group compared with those in the control group. The molecular mechanisms that provide the beneficial effects of periostin were connected with the upregulated expression of integrinβ1/FAK/PI3K/Akt/eNOS signal pathway and the increased secretion of growth factors.ConclusionOverexpression of periostin by gene transfection on ADSCs promotes survival, migration, and therapeutic efficiency, which will bring new insights into the treatment of critical limb ischemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0126-x) contains supplementary material, which is available to authorized users.

Dental pulp stem cells derived conditioned medium promotes angiogenesis in hindlimb ischemia

Paracrine effects of mesenchymal stem cells (MSCs) have been suggested play an important role in the treatment of ischemic diseases. Dental pulp Stem cells (DPSCs) share many properties with MSCs. However, the beneficial effects of DPSCs on ischemic diseases remain to be elucidated. The present study, we found that DPSCs secreted higher levels angiogenic factors of VEGF, SDF-1, MCP-1 and PDGF-BB compared with AD-MSCs. We then investigated whether DP-CM can induce the migration of vascular smooth muscle cells (VSMCs) and human umbilical venous endothelial cells (HUVECs) in vitro. Under hypoxia, the apoptosis of HUVECs was inhibited while survival was improved by treatment of DP-CM. In a H2O2-induced cell death assay, DP-CM also significantly reduced HUVECs oxidative stress compare to control group. The tube formation assay demonstrated that the DP-CM group had a greater angiogenic potential than control medium. Results in the mouse model showed both the laser Doppler perfusion index and the relative number of CD31 positive microvessels to be significantly higher in the DP-CM group than in the control group [(77%±11%) vs. (45%±6%), and (6.2±1.1)/HPF vs. (2.3±0.3)/HPF]. In this way, the use of DP-CM may be a suitable means of treating ischemic diseases.

Sustained Release of Multiple Growth Factors from Injectable Polymeric System as a Novel Therapeutic Approach Towards Angiogenesis

PurposeThe aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion.MethodsThree groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF165 (VEGF), or alginate containing VEGF165 combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points.ResultsIn the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles.ConclusionsSequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.

Abstract 229: The Novel Angiogenic Cytokine Secretoneurin promotes Angiogenesis, Arteriogenesis and Vasculogenesis in the Hind-Limb Ischemia Model

Introduction - Secretoneurin (SN) represents a sensory, inflammatory neuropeptide which was recently demonstrated to act as an angiogenic and vasculogenic cytokine in vitro and in vivo. The present study was conducted to test the hypothesis that SN may be implicated in reparative angiogenesis. Furthermore, we challenged the healing potential of SN applied as a newly generated SN gene therapy vector in the setting of limb ischemia. Methods and Results - We cloned the human SN coding sequence into the pAAV plasmid containing a cytomegalovirus enhancer/promoter sequence. Bioactivity of recombinant SN was shown by proliferative and chemotactic activity on endothelial cells in vitro. Unilateral limb ischemia was induced in C57/bl mice by femoral artery resection. By Real Time PCR, Western Blotting, SN-specific RIA and Immunhistochemistry, we documented that SN is up-regulated in ischemic muscles. Next, we tested whether SN gene therapy may exert curative effects in this ischemia model. Injection of the SN plasmid into ischemic adductor muscles increased capillary (0.67 vs. 0.35, n = 24, p = 0.02) and arteriole (0.16 vs. 0.8, n = 24, p = 0.04) density, reduced endothelial cell apoptosis, and accelerated perfusion recovery as shown by Laser Doppler Perfusion Index (LDPI ratio ischemic/control leg after 28 days of ischemia 1.1 vs. 0.7, n = 24, p &lt; 0.01) in comparson to pAAV-GFP (green-fluorescence protein) treated mice. Furthermore, SN gene therapy significantly reduced toe necrosis of ischemic limbs compared to control animals (26% vs. 50%, n = 24, p &lt; 0.05). In bone marrow transplantation models, increased vascularity of ischemic hind-limbs after SN gene therapy was shown to be mediated, at least in part, by enhanced recruitment of bone marrow-derived endothelial progenitor cells. Conclusions -These results suggest that the novel angiogenic cytokine Secretoneurin is up-regulated by ischemia in skeletal muscle cells. Furthermore, results from gene therapy in this ischemia model suggest that Secretoneurin represent a promising new substance for therapeutic angiogenesis.

This Month in Anesthesiology

This Month in Anesthesiology

Fluorescence Dilution Technique for Measurement of Cardiac Output and Circulating Blood Volume in Healthy Human Subjects

The authors previously validated in an animal model a new indicator dilution technique for measuring cardiac output and circulating blood volume by recording transcutaneously the fluorescence of circulating indocyanine green with an optical probe placed on the skin surface. The current study compared fluorescence dilution recordings recorded from several locations on the human face in terms of signal intensity and stability and estimated the subjects' cardiac output and circulating blood volume from the recordings. Fluorescence dilution traces were recorded transcutaneously in six healthy human volunteers after rapid intravenous injection of 1 mg indocyanine green. Three placements of the optical probe were tested: the nose ala, the ear lobe, and the temple area. In three subjects, the recordings were calibrated in terms of circulating indocyanine green concentration to estimate cardiac output and circulating blood volume. Fluorescence dilution traces had the same duration for the three locations, but the recordings obtained from the nose ala and the ear lobe were twice as intense as those from the temple. The fluorescence intensity at each site was linearly related to the local laser Doppler perfusion index. The coefficient of variation for the area under the first pass curve (inversely proportional to cardiac output) was approximately 6% for triplicate measurements at the same location. Cardiac output and circulating blood volume derived from the fluorescence recordings were in the normal range. The study demonstrates that intense and reproducible fluorescence dilution signals can be measured transcutaneously in healthy humans and could potentially be used to measure cardiac output and circulating blood volume minimally invasively.

Direct Comparison of Human Mesenchymal Stem Cells Derived from Adipose Tissues and Bone Marrow in Mediating Neovascularization in Response to Vascular Ischemia

Background/Aim: Although transplantation of MSC derived from bone marrow or adipose tissues has been shown in proangiogenic action in hindlimb ischemia model of nude mice, little information is available regarding comparison of the angiogenic potency between human adipose stromal cells (hADSC) and bone marrow stromal cells (hBMSC). We compared their therapeutic potential by transplantation of equal numbers of hADSC or hBMSC in a nude mice model of hindlimb ischemia. Methods&Results: One day after creating hindlimb ischemia, mice were randomized to receive hADSC transplantation (hADSC group), hBMSC transplantation (hBMSC group), or vehicle transplantation (Control group). Two weeks after transplantation, the laser Doppler perfusion index was significantly higher in the hADSC group and hBMSC group than in the control group. Comparison between hADSC and hBMSC group showed better recovery of blood flow in hADSC group than in hBMSC group. Conditioned media from hADSC (hADSC-CM) showed better in vitro tube formation of hADSC than conditioned media from hBMSC (hBMSC-CM). hADSC showed higher expression of MMP3 and MMP9 than hBMSC. A MMP inhibitor, GM6001, and the transfection of MMP3 or MMP9 siRNA oligonucleotides inhibited in vitro tube formation of hADSC. Transplantation of MMP3 or MMP9 siRNA oligonucleotieds-transfected hADSC showed lower blood flow recovery and higher tissue injury than control oligonucelotide-transfected cells. Conclusion: This study showed that hADSC can be an ideal source for therapeutic angiogenesis in ischemic disease in terms of efficacy, accessibility and available tissue amounts.

Comparison of angiogenic potency between mesenchymal stem cells and mononuclear cells in a rat model of hindlimb ischemia

Mesenchymal stem cells (MSC) are pluripotent cells that differentiate into a variety of cells including endothelial cells and vascular smooth muscle cells. Although transplantation of bone marrow-derived mononuclear cells (MNC) has already been applied for the treatment of critical limb ischemia, little information is available regarding comparison of the angiogenic potency between MSC and MNC. Accordingly, we injected equal numbers of MSC or MNC in a rat model of hindlimb ischemia and compared their therapeutic potential. Immediately after creating hindlimb ischemia, rats were randomized to receive MSC transplantation (MSC group), MNC transplantation (MNC group), or vehicle infusion (Control group). Three weeks after transplantation, the laser Doppler perfusion index was significantly higher in the MNC group than in the Control group (0.69+/-0.1 vs. 0.57+/-0.06, P<0.01). Furthermore, there was a marked improvement in blood perfusion in the MSC group (0.81+/-0.08). Capillary density was highest in the MSC group. The number of transplanted cell-derived endothelial cells was higher in the MSC group than in the MNC group. Transplanted cell-derived vascular smooth muscle cells were detected only in the MSC group. In vitro, MSC were more tolerant to apoptotic stimulus (serum starvation and hypoxia) than MNC. MSC transplantation caused significantly greater improvement in hindlimb ischemia than MNC transplantation. Compared with MNC, MSC survived well under an ischemic environment, and differentiated into not only endothelial cells but also vascular smooth muscle cells. Thus, MSC transplantation may be a new therapeutic strategy for the treatment of severe peripheral vascular disease.

Adrenomedullin Enhances Angiogenic Potency of Bone Marrow Transplantation in a Rat Model of Hindlimb Ischemia

Previous studies have shown that adrenomedullin (AM) inhibits vascular endothelial cell apoptosis and induces angiogenesis. We investigated whether AM enhances bone marrow cell-induced angiogenesis. Immediately after hindlimb ischemia was created, rats were randomized to receive AM infusion plus bone marrow-derived mononuclear cell (MNC) transplantation (AM+MNC group), AM infusion alone (AM group), MNC transplantation alone (MNC group), or vehicle infusion (control group). The laser Doppler perfusion index was significantly higher in the AM and MNC groups than in the control group (0.74+/-0.11 and 0.69+/-0.07 versus 0.59+/-0.07, respectively, P<0.01), which suggests the angiogenic potency of AM and MNC. Importantly, improvement in blood perfusion was marked in the AM+MNC group (0.84+/-0.08). Capillary density was highest in the AM+MNC group, followed by the AM and MNC groups. In vitro, AM inhibited MNC apoptosis, promoted MNC adhesiveness to a human umbilical vein endothelial cell monolayer, and increased the number of MNC-derived endothelial progenitor cells. In vivo, AM administration not only enhanced the differentiation of MNC into endothelial cells but also produced mature vessels that included smooth muscle cells. A combination of AM infusion and MNC transplantation caused significantly greater improvement in hindlimb ischemia than MNC transplantation alone. This effect may be mediated in part by the angiogenic potency of AM itself and the beneficial effects of AM on the survival, adhesion, and differentiation of transplanted MNCs.

Laser Doppler Perfusion Imaging (LDPI) and Transepidermal Water Loss (TEWL) Values in Psoriatic Lesions Treated with Narrow Band UVB Phototherapy. Dermal Vascularity may be useful Indicator of Psoriatic Activity

Objective: This study attempts to objectively measure physiological changes in transepidermal water loss (TEWL), an indicator of skin barrier function and laser Doppler perfusion index (LDPI) an indicator of skin vascularity, of psoriatic skin lesions following treatment to narrowband ultraviolet B (UVB) phototherapy using the psoriasis severity (PS) score as a measurement of clinical phototherapy response. Materials and Methods: Fourteen patients with established diagnosis of plaque type psoriasis were studied. The patients received narrow band UVB phototherapy 3 times a week until clearance of their psoriasis and the frequencies are reduced as clearance is observed. Two psoriasis plaques (“lesional skin”) were used to measure treatment response and another 2 areas of uninvolved (“non-lesional skin”) on the corresponding opposite limbs were identified for comparison. PS score, TEWL and LDPI were carried out at baseline (0 week), 2 weeks and 8 weeks during treatment. Measurements were carried out just before each phototherapy session and repeated 1 hour after phototherapy treatment. Results: The mean PS score decreased by almost 40% after 2 months phototherapy (t = 2.44, P = 0.028). There was no significant difference in PS score between week 0 and week 2, or between week 2 and week 8. After phototherapy, there was no significant difference in LDPI values on the psoriatic lesional skin between week 0 and week 8 or between week 2 and week 8. It appears that phototherapy does not induce inflammation on non-lesional skin to provoke measurable LDPI changes. The mean TEWL values of psoriatic lesional skin were significantly higher than normal skin throughout the study period before and during phototherapy (week 0, t = 5.71, P = 0.000, week 2, t = 9.29, P = 0.00, week 8, t = 6.93, P = 0.000). It appears that the skin barrier function of psoriatic skin improves only minimally as psoriatic lesions improve clinically (as evidenced by the reduction in PS scores) with narrow-band UVB phototherapy, but the dermal vascularity and blood flow and barrier function of the psoriatic skin remained abnormal. Conclusion: It appears that LDPI and TEWL measurement may not be a good surrogate marker of clinical response to narrow-band UVB phototherapy.