A New Approach to Vascular Screening: Identification of Impaired Vascular Function Using the FMSF Technique.

Arterial blood pressure monitoring plays an important role in preventive medicine, allowing, in selected cases, the identification of vascular dysfunction. In this review, we propose a new non-invasive approach to assessment of the circulatory system, based on its reaction to hypoxia induced by post-occlusive reactive hyperemia (PORH). Three key parameters can be used for vascular screening: the Reactive Hyperemia Response (RHR), which represents the overall reaction of the macro- and microcirculation to transient hypoxia; Hypoxia Sensitivity (HS), which reflects hypoxia-induced activation of myogenic oscillations of the microcirculation; and Normoxia Oscillatory Index (NOI), which characterizes microcirculatory oscillations under normoxia conditions. A method for assessing these parameters, analogous in simplicity to arterial blood pressure measurement, is provided by the Flow Mediated Skin Fluorescence (FMSF) technique. Reference values are proposed based on numerous test measurements.

Assessment of NADH/NAD+ Redox Imbalance in Psoriatic Lesions Using the FMSF Technique: Therapeutic Aspects.

Mitochondrial dysfunction has been linked to psoriasis, and it may be an important underlying factor contributing to this disease. However, a precise methodology for assessing mitochondrial dysfunction has yet to be developed. One promising approach is to measure NADH autofluorescence from the affected skin areas. In this study, we show that Flow-Mediated Skin Fluorescence (FMSF) can be used for the non-invasive assessment of mitochondrial dysfunction in psoriasis. The fluorescence level at baseline and the half-time of ischemic growth (t1/2) derived from the FMSF traces can be used for the non-invasive assessment of NADH/NAD+ redox imbalance in psoriatic lesions compared to unaffected skin. These results are supported by an analysis of the key FMSF parameters: Reactive Hyperemia Response (RHR) and Hypoxia Sensitivity (HS). This method not only contributes to understanding the biochemical processes involved in the etiopathogenesis of psoriasis, but it also provides a basis for identifying new drug targets and improving the treatment process.

Boronate-Based Oxidant-Responsive Derivatives of Acetaminophen as Proinhibitors of Myeloperoxidase.

Myeloperoxidase (MPO) is an important component of the human innate immune system and the main source of a strong oxidizing and chlorinating species, hypochlorous acid (HOCl). Inadvertent, misplaced, or excessive generation of HOCl by MPO is associated with multiple human inflammatory diseases. Therefore, there is a considerable interest in the development of MPO inhibitors. Here, we report the synthesis and characterization of a boronobenzyl derivative of acetaminophen (AMBB), which can function as a proinhibitor of MPO and release acetaminophen, the inhibitor of chlorination cycle of MPO, in the presence of inflammatory oxidants, i.e., hydrogen peroxide, hypochlorous acid, or peroxynitrite. We demonstrate that the AMBB proinhibitor undergoes conversion to acetaminophen by all three oxidants, with the involvement of the primary phenolic product intermediate, with relatively long half-life at pH 7.4. The determined rate constants of the reaction of the AMBB proinhibitor with hydrogen peroxide, hypochlorous acid, or peroxynitrite are equal to 1.67, 1.6 × 104, and 1.0 × 106 M-1 s-1, respectively. AMBB showed lower MPO inhibitory activity (IC50 > 0.3 mM) than acetaminophen (IC50 = 0.14 mM) toward MPO-dependent HOCl generation. Finally, based on the determined reaction kinetics and the observed inhibitory effects of two plasma components, uric acid and albumin, on the extent of AMBB oxidation by ONOO- and HOCl, we conclude that ONOO- is the most likely potential activator of AMBB in human plasma.

Non-Invasive Assessment of Vascular Circulation Based on Flow Mediated Skin Fluorescence (FMSF).

Flow Mediated Skin Fluorescence (FMSF) is a new non-invasive method for assessing vascular circulation and/or metabolic regulation. It enables assessment of both vasoconstriction and vasodilation. The method measures stimulation of the circulation in response to post-occlusive reactive hyperemia (PORH). It analyzes the dynamical changes in the emission of NADH fluorescence from skin tissue, providing the information on mitochondrial metabolic status and intracellular oxygen delivery through the circulatory system. Assessment of the vascular state using the FMSF technique is based on three parameters: reactive hyperemia response (RHR), hypoxia sensitivity (HS), and normoxia oscillatory index (NOI). The RHR and HS parameters determine the risk of vascular circulatory disorders and are the main diagnostic parameters. The NOI parameter is an auxiliary parameter for evaluating the state of microcirculation under stress of various origins (e.g., emotional stress, physical exhaustion, or post-infection stress). The clinical data show that the risk of vascular complications is limited among people whose RHR, log(HS), and NOI parameters are not significantly below the mean values determined by the FMSF technique, especially if they simultaneously meet the conditions RHR > 30% and log(HS) > 1.5 (HS > 30), and NOI > 60%.

Chronic Fatigue Associated with Post-COVID Syndrome versus Transient Fatigue Caused by High-Intensity Exercise: Are They Comparable in Terms of Vascular Effects?

Purpose: The pathophysiology of chronic fatigue associated with post-COVID syndrome is not well recognized. It is assumed that this condition is partly due to vascular dysfunction developed during an acute phase of infection. There is great demand for a diagnostic tool that is able to clinically assess post-COVID syndrome and monitor the rehabilitation process. Patients and Methods: The Flow Mediated Skin Fluorescence (FMSF) technique appears uniquely suitable for the analysis of basal microcirculatory oscillations and reactive hyperemia induced by transient ischemia. The FMSF was used to measure vascular circulation in 45 patients with post-COVID syndrome. The results were compared with those for a group of 26 amateur runners before and after high-intensity exercise as well as for a control group of 32 healthy age-matched individuals. Results: Based on the observed changes in the NOI (Normoxia Oscillatory Index) and RHR (Reactive Hyperemia Response) parameters measured with the FMSF technique, it was found that chronic fatigue associated with post-COVID syndrome is comparable with transient fatigue caused by high-intensity exercise in terms of vascular effects, which are associated with vascular stress in the macrocirculation and microcirculation. Acute and chronic fatigue symptomatology shared similarly altered changes in the NOI and RHR parameters and both can be linked to calcium homeostasis modification. Conclusion: The NOI and RHR parameters measured with the FMSF technique can be used for non-invasive clinical assessment of post-COVID syndrome as well as for monitoring the rehabilitation process.

Fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid: a comparative study.

MPO-derived oxidants including HOCl contribute to tissue damage and the initiation and propagation of inflammatory diseases. The search for small molecule inhibitors of myeloperoxidase, as molecular tools and potential drugs, requires the application of high throughput screening assays based on monitoring the activity of myeloperoxidase. In this study, we have compared three classes of fluorescent probes for monitoring myeloperoxidase-derived hypochlorous acid, including boronate-, aminophenyl- and thiol-based fluorogenic probes and we show that all three classes of probes are suitable for this purpose. However, probes based on the coumarin fluorophore turned out to be not reliable indicators of the inhibitors' potency. We have also determined the rate constants of the reaction between HOCl and the probes and they are equal to 1.8 × 104 M-1s-1 for coumarin boronic acid (CBA), 1.1 × 104 M-1s-1 for fluorescein based boronic acid (FLBA), 3.1 × 104 M-1s-1 for 7-(p-aminophenyl)-coumarin (APC), 1.6 × 104 M-1s-1 for 3'-(p-aminophenyl)-fluorescein (APF), and 1 × 107 M-1s-1 for 4-thiomorpholino-7-nitrobenz-2-oxa-1,3-diazole (NBD-TM). The high reaction rate constant of NBD-TM with HOCl makes this probe the most reliable tool to monitor HOCl formation in the presence of compounds showing HOCl-scavenging activity.

A derivative of vitamin B3 applied several days after exposure reduces lethality of severely irradiated mice.

Most, if not all, of the hitherto tested substances exert more or less pronounced pro-survival effects when applied before or immediately after the exposure to high doses of ionizing radiation. In the present study we demonstrate for the first time that 1-methyl nicotinamide (MNA), a derivative of vitamin B3, significantly (1.6 to 1.9 times) prolonged survival of BALB/c mice irradiated at LD30/30 (6.5 Gy), LD50/30 (7.0 Gy) or LD80/30 (7.5 Gy) of γ-rays when the MNA administration started as late as 7 days post irradiation. A slightly less efficient and only after the highest dose (7.5 Gy) of γ-rays was another vitamin B3 derivative, 1-methyl-3-acetylpyridine (1,3-MAP) (1.4-fold prolonged survival). These pro-survival effects did not seem to be mediated by stimulation of haematopoiesis, but might be related to anti-inflammatory and/or anti-thrombotic properties of the vitamin B3 derivatives. Our results show that MNA may represent a prototype of a radioremedial agent capable of mitigating the severity and/or progression of radiation-induced injuries when applied several hours or days after exposure to high doses of ionizing radiation.

Differentiation of Diabetic Foot Ulcers Based on Stimulation of Myogenic Oscillations by Transient Ischemia.

PURPOSE: Diabetic foot ulceration is a chronic complication characterized by impaired wound healing. There is a great demand for a diagnostic tool that is able to monitor and predict wound healing. PATIENTS AND METHODS: Oscillations in the microcirculation, known as flowmotion, can be monitored very distinctly and precisely using the Flow Mediated Skin Fluorescence (FMSF) technique. The flowmotion response to hypoxia was measured quantitatively in 42 patients with diabetic foot ulcers. RESULTS: The flowmotion response to hypoxia parameters FM(R) and HS were used to differentiate the diabetic foot ulcers and correlate them with clinical status. In some cases, FMSF measurements were continued over the period of a year in order to monitor disease progress. The clinical status of the quarter of patients with the highest HS values (group A, HS = 50.2±18.3) was compared to the quarter with the lowest HS values (group B, HS = 4.3±1.7). The patients in the group B were identified as having low prognosis for healing and were characterized by higher incidences of hypertension, hyperlipidemia, prevalent CVD, neuropathy and nephropathy. CONCLUSION: Impaired flowmotion responses to hypoxia induced by transient ischemia can be used for differentiation of diabetic foot ulcers and identification of cases with low prognosis for healing.

Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide.

Hydroethidine (HE) and hydropropidine ([Formula: see text]) are fluorogenic probes used for the detection of the intra- and extracellular superoxide radical anion ([Formula: see text]). In this study, we provide evidence that HE and [Formula: see text] react rapidly with the biologically relevant radicals, including the hydroxyl radical, peroxyl radicals, the trioxidocarbonate radical anion, nitrogen dioxide, and the glutathionyl radical, via one-electron oxidation, forming the corresponding radical cations. At physiological pH, the radical cations of the probes react rapidly with [Formula: see text], leading to the specific 2-hydroxylated cationic products. We determined the rate constants of the reaction between [Formula: see text] and the radical cations of the probes. We also synthesized N-methylated analogs of [Formula: see text] and HE which were used in mechanistic studies. Methylation of the amine groups was not found to prevent the reaction between the radical cation of the probe and the superoxide, but it significantly increased the lifetime of the radical cation and had a substantial effect on the profiles of the oxidation products by inhibiting the formation of dimeric products. We conclude that the N-methylated analogs of HE and [Formula: see text] may be used as a scaffold for the design of a new generation of probes for intra- and extracellular superoxide.

Flowmotion Monitored by Flow Mediated Skin Fluorescence (FMSF): A Tool for Characterization of Microcirculatory Status.

Oscillations in the microcirculation, known as flowmotion, are a well-recognized characteristic of cutaneous blood flow. Since flowmotion reflects the microcirculatory status of the vascular system, which is very often impaired in many diseases and disorders, a quantitative assessment of skin flowmotion could potentially be used to screen for early symptoms of such conditions. In this study, skin flowmotion was monitored using the Flow Mediated Skin Fluorescence (FMSF) technique. The flowmotion parameter was used for quantitative assessment of basal flowmotion both at rest (FM) and during reperfusion [FM(R)] following the post-occlusive reactive hyperemia (PORH). The study population was composed of healthy volunteers between the ages of 30 and 72 (n = 75). The FM parameter showed an inverse dependence relative to age, while the FM(R) parameter was inversely correlated to blood pressure. The FM(R) parameter reflects the strong effect of hypoxia on flowmotion, which is mainly due to increased myogenic activity in the vessels. The FMSF technique appears to be uniquely suited for the analysis of basal flowmotion and the hypoxia response, and may be used for the characterization of microcirculatory status.

4-Methylpseudoproline analogues of cyclolinopeptide A: Synthesis, structural analysis and evaluation of their suppressive effects in selected immunological assays.

The synthesis of new analogues of cyclolinopeptide A (CLA) and their linear precursors modified with (R)- and (S)-4-methylpseudoproline in the Pro3-Pro4 fragment are presented. The peptides were tested in comparison with cyclosporine A (CsA) in concanavalin A (Con A) and pokeweed mitogen (PWM)-induced mouse splenocyte proliferation and in secondary humoral immune response in vitro to sheep erythrocytes (SRBC). Their effects on expression of selected signaling molecules in the Jurkat T cell line were also determined. In addition, the structural features of the peptides, applying nuclear magnetic resonance and circular dichroism, were analyzed. The results showed that only peptides 7 and 8 modified with (R)-4-methylpseudoproline residue (c(Leu1-Val2-(R)-(αMe)Ser(ΨPro)3-Pro4-Phe5-Phe6-Leu7-Ile8-Ile9) and c(Leu1-Val2-Pro3-(R)-(αMe)Ser(ΨPro)4-Phe5-Phe6-Leu7-Ile8-Ile9), respectively) strongly suppressed mitogen-induced splenocyte proliferation and the humoral immune response, with peptide 8 being more potent. Likewise, peptide 8 more strongly elevated expression of Fas, a proapoptotic signaling molecule in Jurkat cells. We postulate that the increased biological activity of peptide 8, compared to the parent molecule and other studied peptides, resulted from its more flexible structure, found on the basis of both CD and NMR studies. CD and NMR spectra showed that replacement of Pro3 by (R)-(αMe)Ser(¬Pro) caused much greater conformational changes than the same replacement of the Pro4 residue. Such a modification could lead to increased conformational freedom of peptide 8, resulting in a greater ability to adopt a more compact structure, better suited to its putative receptor. In conclusion, peptide 8 is a potent immune suppressor which may find application in controlling immune disorders.

Flow Mediated Skin Fluorescence technique reveals remarkable effect of age on microcirculation and metabolic regulation in type 1 diabetes.

STUDY DESCRIPTION: Flow Mediated Skin Fluorescence (FMSF) is a novel technique for non-invasive evaluation of the microcirculation and metabolic regulation. This study describes the diagnostic potential of FMSF for type 1 diabetes (DM1). STUDY POPULATION: All study participants, in both the control (n = 31) and DM1 (n = 40) groups, were between the ages of 30-49 y. The patients in the DM1 group had all been suffering from diabetes for at least 10 y. RESULTS: The parameters HRindex, HRmax and MR inversely correlate with age and BMI. An unidentified compensatory effect was observed among the younger members of the DM1 group. The majority of DM1 patients with HRindex < 8% showed signs of dysfunctional metabolic regulation. CONCLUSION: FMSF appears to be an extremely useful technique for monitoring diabetic patients over time, enabling early diagnosis of potentially dysfunctional microcirculation and metabolic regulation.

Decomposition of Piloty's acid derivatives - Toward the understanding of factors controlling HNO release.

The recent interest in the clinical applications of Piloty's acid derivatives as HNO donors for the treatment of cardiovascular system dysfunction has led us to the examination of factors controlling HNO release from selected ortho-substituted N-hydroxysulfonamides. Here we present the kinetic and quantum mechanical studies on the mechanism of HNO release from selected ortho-substituted N-hydroxysulfonamides and in vivo examination of the antiaggregatory properties of N-hydroxy-(2-bromobenzene)sulfonamide complex with sodium salt of ß-cyclodextrin sulfobutyl ethers-ethyl ethers as compared with Angeli's salt.

Fluorescent probes for the detection of nitroxyl (HNO).

Nitroxyl (HNO), which according to the IUPAC recommended nomenclature should be named azanone, is the protonated one-electron reduction product of nitric oxide. Recently, it has gained a considerable attention due to the interesting pharmacological effects of its donors. Although there has been great progress in the understanding of HNO chemistry and chemical biology, it still remains the most elusive reactive nitrogen species, and its selective detection is a real challenge. The development of reliable methodologies for the direct detection of azanone is essential for the understanding of important signaling properties of this reactive intermediate and its pharmacological potential. Over the last decade, there has been considerable progress in the development of low-molecular-weight fluorogenic probes for the detection of HNO, and therefore, in this review, we have focused on the challenges and limitations of and perspectives on nitroxyl detection based on the use of such probes.