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.

Conclusions from an Observational Study of Patients with Vascular Diseases Using the FMSF Technique.

Purpose: There is great demand for a diagnostic tool for non-invasive assessment of vascular circulation and metabolic regulation. Assessing both these functions is crucial, as each can have a distinct response to hypoxia. Patients and Methods: The Flow Mediated Skin Fluorescence (FMSF) technique appears uniquely suitable for analysis of vascular circulation and metabolic regulation. In this observational study, the FMSF technique was used to diagnose patients with various vascular diseases. The study group consisted of 482 patients (264 females and 218 males) between the ages of 40-94 years with various vascular problems (arterial hypertension, cardiovascular disease, diabetes, hypercholesterolemia, and chronic venous disease). Results: Three major FMSF parameters were used: Ischemic Response (IRmax), Hyperemic Response (HRmax), and Reactive Hyperemia Response (RHR). All three parameters were found to decrease with age with a distinguishable kinetics. The IRmax parameter was used for characterization of metabolic reaction to transient hypoxia and HRmax was used for characterization of macrocirculatory function. Both were sex-dependent. Conclusion: Females were metabolically less adaptive to transient hypoxia than males. However, macrocirculatory function was better in females than among males. Microcirculatory function decreases gradually with age, while macrocirculatory function decreases much more slowly with age, with a tendency to stabilize after 70 years of age.

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.

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.

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.

4-Methylpseudoproline derived from α-methylserine - synthesis and conformational studies.

This paper presents the synthesis and solution conformational studies of the tripeptides Fmoc-Ala-(R)-(αMe)Ser(Ψ(H,H)Pro)-Ala-OBu(t) (6a) and Fmoc-Ala-(S)-(αMe)Ser(Ψ(H,H)Pro)-Ala-OBu(t) (6b). Additionally, the X-ray structure of 6a is given. NMR analysis corroborated by theoretical calculations (XPLOR) shows that in both peptides the amide bond between pseudoproline and the preceding amino acid is in the trans conformation. The same amide bond geometry was observed in the crystal state of 6a. The latter is additionally influenced by the presence of two symmetrically independent molecules in an asymmetric unit. Both molecules adopt a conformation which resembles ß-turn type II, stabilized by hydrogen bonding. The conformational preferences and prolyl cis-trans isomerization of Ac-(αMe)Ser(Ψ(H,H)Pro)-NHMe (7) were explored at the IEFPCM/B3LYP/6-31+G(d) level of theory in vacuum, water and chloroform. It has been shown that the trans isomer predominates in water solutions and the cis isomer is preferred in chloroform. The conformation of 7 is down-puckered independently of the geometry of the amide bonds, with lower puckering in the transition state of the cis-trans isomerization.

Cyclolinopeptide derivatives modify methotrexate-induced suppression of the humoral immune response in mice.

High doses of chemotherapeutics in clinical treatment, leading to cell toxicity, can be lowered by co-administration of other immunoregulatory drugs. The aim of this study was to evaluate effects of several derivatives of cyclolinopeptide A (CLA), derived from linen seeds, on the suppressive action of metothrexate (MTX) in a mouse model of humoral immune response in vitro. New CLA analogues 1 and 2, and their linear precursors 3 and 4, containing conformationally restricted dipeptide fragment Phe-Phe or D-Phe-D-Phe with ethylene bridge (-CH(2)-CH(2)-) between phenylalanine nitrogens were synthesized. NMR studies and theoretical calculations showed that introduction of locally constraining fragment into CLA molecule increased its overall conformational flexibility. The bioactivity of new CLA analogues was examined in the mouse model of the in vitro secondary humoral immune response, suppressed by methotrexate (MTX). The results revealed differential actions of the peptides such as 1/augmentation of the suppressive activity of MTX or 2/antagonistic effects of the peptides on MTX-induced suppression. Potential advantages for the application of CLA-derived peptides in therapy and structure-activity relationships were discussed.

New bradykinin B(2) receptor antagonists - influence of C-terminal segment modifications on their pharmacological properties.

In the present study we describe the synthesis and some pharmacological properties of eight new analogues of bradykinin (BK). Two peptides were designed by substitution of position 7 or 8 of the known [D-Arg(0),Hyp(3),Thi(5,8),D-Phe(7)]BK antagonist (Stewart's antagonist) with L-pipecolic acid (L-Pip). The next two analogues were obtained by replacement of the d-Phe residue in position 7 of the Stewart's peptide with L-beta(2)-isoproline (L-beta(2)-iPro) or L-beta(3)-homoproline (L-beta(3)-hPro). The four analogues mentioned above were also prepared in N-acylated form with 1-adamantaneacetic acid (Aaa). Biological activity of the compounds was assessed by isolated rat uterus and rat blood pressure tests. Our results showed that L-Pip in position 7 slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. Replacement of Thi by L-Pip in position 8 also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. L-beta(2)-iPro or L-beta(3)-hPro in position 7 decreased the potencies in both tests. We also demonstrated that acylation of the N-terminus did not increase, as was claimed previously, the antagonistic potencies of the resulting peptides. The results thus support the hypothesis about the existence of different types of BK receptors in the rat uterus and blood vessels. Our studies provide new information about the structure-activity relationship of BK antagonists which may help in designing more potent BK receptor blockers.

Synthesis and immunosuppressive activity of new cyclolinopeptide A analogs modified with beta-prolines.

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. As a continuation of our previous work searching for new, effective suppressors devoid of toxicity, we present the synthesis, conformational analysis, and biological activity of nonapeptides 1-6, analogs of naturally existing immunomodulatory peptide CLA. New CLA analogs were modified with (S)-beta(2)-iso-proline 7 or (S)-beta(3)-homo-proline 8, respectively. The conformational influence of the beta-iso-proline and beta-homo-proline building blocks was analyzed by NMR spectroscopy. Peptides 1-6 exist as a mixture of four isomers due to cis/trans isomerization of the Xxx-Pro peptide bond. The major isomers of peptides 1, 3, and 4 contain all peptide bonds of the trans geometry. The geometry of the proline-proline bond of the second populated isomer of peptides 3 and 4 is cis. The proline-proline peptide bond is cis for the major isomers of peptides 2, 5, and 6. The peptides were tested for their ability to suppress the proliferative response of mouse splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep erythrocytes in vitro in parallel with a reference drug-cyclosporine A. The immunoregulatory actions of the peptides depended on the position and content of proline isomers and were, with some exceptions, strongly inhibitory at the highest dose tested (100 microg/ml). In addition, the peptides were practically devoid of toxicity at that dose. In conclusion, the replacement of Pro by beta-Pro may be useful for fine-tuning CLA immunosuppressive potency and undesirable toxicity.