Studies on the Adaptive Responses to Hypoxia
Studies on the Adaptive Responses to Hypoxia
The general aim of this study is to define the response to hypoxic challenge in patients with
 diabetes. The investigation will provide response for different questions that are central
 for explaining the development of complications in diabetes

 -  have patients with diabetes an impaired reaction to adapt to hypoxia

 -  what consequence has hypoxia challenge on respiratory and on cardiovascular regulation
 in patients with diabetes

 -  what consequence has diabetes on the angiogenetic response to hypoxia
Complications of diabetes represent the main concern for modern diabetes therapy, and it has
 become a priority to further characterise the pathophysiological mechanisms of these
 complications to ensure the development of novel rational therapeutic strategies.

 Although the prolonged exposure of tissues to hyperglycaemia is the primary causative factor
 for chronic diabetes complications, it has recently become increasingly evident that hypoxia
 also plays an important role in all diabetes complications . A low tissue concentration of
 oxygen in diabetes is the consequence of several mechanisms (e.g., deficient blood supply
 because of micro- and macro-vascular disease , poor local oxygen diffusion because of local
 oedema, or as the result of increased oxygen consumption).

 Adaptive responses of cells to hypoxia are mediated by Hypoxia-Inducible Factor 1 (HIF),
 which is a heterodimeric transcription factor that is composed of and subunits, which are
 both constitutively expressed in mammalian cells. The regulation of HIF activity is
 critically dependent on subunit degradation in normoxia. Under hypoxic conditions, HIF-1 is
 stabilised, binds to HRE (hypoxic responsive elements) and up-regulates a gene series that is
 involved in angiogenesis, glycolytic energy metabolism, cell proliferation, and survival,
 which enables cells to adapt to reduced oxygen availability. It is estimated that that more
 than 800 genes are direct HIF targets . HIF-1 is central for expression of multiple
 angiogenic growth factors (reviewed by[6]), endothelial progenitor cells (EPC) recruitment .
 Recently, it has been proposed that microRNAs (ex. mir210) also mediate some HIF-1 functions
 .

 Several pieces of evidence point to a defective response of diabetic tissues to hypoxia. An
 impaired hypoxia response is present in all tissues investigated in diabetic animal and
 diabetic patients. Hyperglycaemia directly represses HIF stability and function at multiple
 levels, a mechanism that is not completely understood.

 An impaired reaction to hypoxia in diabetes might have important consequences in acute
 hypoxic challenges as acute heart infarction, stroke, limb ischemia but also in subtle
 regulation of cardiovascular and respiratory system as a consequence of autonomic neuropathy
 with potential severe prognostic effect on late cardiovascular events . Interventional
 studies have addressed the cardiovascular responses to intermittent hypoxia (IH) compared
 with normoxia exposure in patients with diabetes . However in order to establish the
 appropriateness of the cardiovascular reaction to hypoxia in diabetes it is a need to
 investigate the cardiorespiratory responses towards IH in patients with diabetes compared
 with matched non diabetic control subjects. Moreover it is essentially to establish the
 angiogenetic response in the same experimental design. The hypothesis for this study is that
 both the cardiorespiratory reaction mediated through autonomic nervous system and
 angiogenetic response are impaired in patients with diabetes as a consequence of an impaired
 HIF reaction as seen in other tissues .

 The investigators plan to study the effect of intermittent hypoxia (IH) that will consists of
 five hypoxic periods (13% O2 inspired fraction of oxygen) each lasting 6 min, with five
 normoxic intervals of same duration in 15 patients with type 1 diabetes without clinical
 signs of neuropathy and in 15 non diabetic controls matched for sex, BMI, age. The study will
 be performed during one day.

 Methods:

 Baseline data will be obtained in the morning of each testing day at least 2 h after
 breakfast. Subjects are advised to abstain from caffeinated beverages for 12 h and from
 alcohol for 36 h prior to testing.

 Blood pressure and heart rate and arterial oxygen saturation will be continuously measured.
 In case of a decrease in oxygen saturation 80% or the occurrence of symptoms, hypoxia would
 have been discontinued until oxygen levels reached at least 80%. A technician will regulate
 and control the breathing periods under supervision of a medical doctor in a way that the
 intervention could not be observed by any patient. Thereafter, three measurement sessions
 will be performed: immediately after (t2), after 3 h (t3), and after 6 h (t4). After t2, each
 patient will obtaine an individual meal according to diet requirement.

 Cardiovascular and respiratory testing.

 Hypoxic ventilatory response (HVR) and hypercapnic ventilatory response (HCVR) will be
 evaluated to determine respiratory system activity. All patients will be tested in the supine
 position in a silent room at comfortable temperature. Before participants will be connected
 to a rebreathing circuit through a mouthpiece with an antibacterial filter, spontaneous
 breathing of room air at rest will be performed for4 min in order to obtain baseline data.

 During each condition, the investigators will perform continuous measurement of oxygen
 saturation (SaO2) by a pulse oximeter and end-tidal CO2 (CO2-et) using a capnograph connected
 to a mouthpiece. Recordings of electrocardiogram will be performed by chest leads, and
 continuous noninvasive blood pressure will be recorded using the cuff method. A heated Fleish
 pneumotachograph will be connected to a differential pressure transducer and inserted in
 series to the expiratory component of the rebreathing system to measure airway flow. For
 measurement of the response to hypoxia, the participants will be connected to a rebreathing
 circuit inducing progressive decrease in SaO2 while maintaining CO2-et values at constant
 levels, until SaO2 will reach 80%, and measuring breath-to-breath changes in minute
 ventilation. The response to hypercapnia will be evaluated by ventilatory changes induced by
 progressive increase in CO2-et levels (up to 13 mmHg above resting levels), while SaO2 will
 be maintained 98% by oxygen at very low flow.

 Cardiovascular autonomic function will be determined performing four tests according to
 recent guidelines: deep-breathing, 30:15 ratio, Valsalva maneuver and systolic blood pressure
 response to standing. Cardiovascular autonomic neuropathy will be defined as the "presence of
 two or more abnormal tests".

 Measurement of chemoreflex sensitivity: The slope of the linear regression line of minute
 ventilation versus SaO2 or CO2-et indicates in each case the chemoreflex sensitivity to
 hypoxia or hypercapnia. In the hypercapnic test, the point at which the ventilation start to
 increase indicates as ventilator recruitment threshold to CO2 (VRT- CO2). VRT- CO2 will be
 identified by interpolating the ventilation/CO2-et plot by a fourth-order polynomial
 function.

 Assessment of baroreflex sensitivity: The baroreflex sensitivity (BRS) will be measured
 during spontaneous breathing at each measurement session. Since previous studies did not
 document a better performance of one method over the others, the investigators will calculate
 the average of seven different methods: positive and negative sequences, the a-coefficient in
 the low- and high-frequency bands and its average, the transfer function technique, and the
 ratio of SDs of R-R interval and systolic blood pressure variabilities. Besides BRS, SD of
 the R-R interval (SDNN) will be applied to determine a global index of heart rate
 variability. This selection is done based on the fact that normal distribution is more
 pronounced in this variable compared with other indices of variability (e.g., variance). The
 Hypoxic ventilatory response (HVR) and hypercapnic ventilatory response (HCVR), baroreflex
 sensitivity (BRS) will be evaluated before (t1), immediately after (t2), 3 h (t3), and 6 h
 (t4) after IH.

 The angiogenetic potential will be evaluated at the same endpoints by measuring in serum
 relevant cytokines that are gene targets for HIF-1 (i.e. VEGFA, SDF-1a, erythropoietin etc).
 The direct response of HIF signaling will be evaluated by the serum levels of mir210 which
 exclusively regulated by HIF.

 The absolute endothelial precoursor cell account (EPC) response will be evaluated at the same
 time points by FACS analysis of the number of CD34+/CD133+/KDR +
Inclusion Criteria:

 1. Patients with typ1 diabetes with a duration of the disease between 10-20 years (HbA1c
 ≥ 65 mmol /l)

 2. Healthy controls matched for age, BMI and gender on group bases.

 Exclusion Criteria:

 1. Smoking

 2. Infections during the last month

 3. Major cardiovascular complications such as coronary heart disease, unstable or stable
 angina, myocardial infarction, ventricular arrhythmias, and atrial fibrillation in the
 last 3 months

 4. Decompensated congestive heart failure or functional class 3-4.

 5. Therapy with b-blockers

 6. Severe hypertension (180 mmHg systolic or 110 mmHg diastolic blood pressure

 7. Proliferative retinopathy.

 8. Obvious sign for diabetic neuropathy (decreased/absent sensitivity to 10 g
 monofilament, vibration, plantar reflex)

 9. Definite autonomic dysfunction

 10. HbA1c ≥ 108 mmol/l

 11. Any concomitant disease or condition that may interfere with the possibility for the
 patient to comply with or complete the study protocol

 12. Malignancy

 13. History of alcohol or drug abuse

 14. Participant in another ongoing pharmacological study

 15. Unwillingness to participate following oral and written information

 16. Subjects with any other severe acute or chronic medical or psychiatric condition that
 make the subject inappropriate for the study in the judgment of the investigator