Cellular and Molecular Mechanisms of Diabetes-Induced Vascular Complications

Question 1

Type 2 Diabetes:

Answer 1

• a developed lack of sensitivity to Insulin
• Risk factors include: genetic predisposition, obesity, unhealthy diet, ethnicity e.g South East Asian, Lack of exercise (often T2DM risk factors are modifiable)

Question 2

What is the UK prevalence of T2DM?

Answer 2

There are currently 3.2 million UK citizens with T2DM- that is 1 in 17 people and uses up 10% of the NHS budget

Question 3

What is Pre-diabetes?

Answer 3

The stage in which insulin sensitivity is decreasing but the body is able to compensate by increasing the production of insulin. Because the body is compensating, diabetes cannot be detected using blood glucose measurements because glucose levels are normal
Pre-diabetes can last for as long as 10 years

Question 4

How does the body compensate in pre-diabetes?

Answer 4

It increases Beta cell mass by increasing cell size and producing more beta cells
It produces more insulin
It increases the production of proinsulin biosynthesis

Question 5

What happens when the body is no longer able to compensate?

Answer 5

The body can no longer overcome the insensitivity to insulin and so full DIABETES ensues.
Beta cell mass decreases and the production of insulin falls. This makes the situation worse because any insulin sensitivity that was left is now useless because less and less insulin is produced

Question 6

What is the problem with pre-diabetes?

Answer 6

It is now thought that much of the damage that causes Cardiovascular complications of diabetes occur during pre-diabetes and because pre-diabetes goes unnoticed, the damage is not being prevented
-It is estimated that 50% of diabetes patients already have evidence of complications at diagnosis

Question 7

Can the damage caused by pre-diabetes not just be solved by good glycaemic control?

Answer 7

NO, evidence has shown that even if good glycaemic control is achieved once diabetes is diagnosed, the damage done in pre-diabetes cannot be reversed and does not improve cardiovascular outcomes

Question 8

Does good glycaemic control improve anything?

Answer 8

Good control of glucose levels improves microvascular complications such as retinopathy, neuropathy and nephropathy, but it makes no difference to macrovascular complications such as Atherosclerosis, stroke, MI etc

Question 9

What is the risk with diabetes?

Answer 9

Diabetics show a risk of mortality from cardiovascular disease that is 2-fold higher than non-diabetics
Diabetes is shown to reduce life expectancy by at least 10 years

Question 10

What is vascular endothelium?

Answer 10

It is the thin layer of cells on the inside of a blood vessel. It acts as the interface between the blood and the vessel and is key to maintaining vascular health

Question 11

What are the functions of healthy endothelium?

Answer 11

• Anti-thrombosis
• Anti-coagulant
• Anti-hypertrophic
• Responsible for Endothelium-Dependent Vasodilation
• Anti-inflammatory

Question 12

Where do smooth muscle cells come in?

Answer 12

A layer of smooth muscle cells sit under the endothelium.
In health these cells are quiescent and demonstrate slow proliferation.
In health these cells are merely contractile to modulate vessel resistance and control blood pressure

Question 13

How is Nitric Oxide related to the vascular endothelium?

Answer 13

The endothelium produces Nitric Oxide.
This is a very important molecule needed for:
1. Reducing oxidation of LDL cholesterol
2. A vasodilator to dilate blood vessels
3. Reducing platelet stickiness (reduces coagulation)
4. Reducing monocyte stickiness (prevents monocytes from sticking to atherosclerotic plaques)
5. Reducing proliferation of smooth muscle cells (keeps the smooth muscle cells quiescent in health)
6. Reducing the release of Superoxide free radicals that may cause damage to cells and structures (can damage DNA)
*When the endothelium is damaged, it can no longer produce NO so none of the above tasks can be completed

Question 14

How is insulin used in healthy cell signalling?

Answer 14

Insulin usually induces the P13K pathway in endothelial cells
Activation of this pathway results in NO and prostaglandin release which causes vasodilation of vascular smooth muscle

Question 15

How does diabetes affect insulin signalling?

Answer 15

Insulin resistance causes an impairment to the P13K pathway and so the MAPK pathway is initiated instead
The MAPK pathway produces Thromboxane which is a vasoconstrictor and platelet aggregator, and also Endothelin 1 (ET-1) which is too a vasoconstrictor

Question 16

How does the endothelium maintain vascular homeostasis?

Answer 16

In health, the endothelium produces more protective factors than harmful:
PROTECTIVE:
1. NO
2. EDHF (Endothelium Derived Hyperpolarising Factor)
3. Prostacyclin (anticoagulant and vasodilator)
4. Bradykinin (Vasodilator)

HARMFUL:

1. Endothelin 1 (ET-1, a vasoconstrictor)
2. ROS (reactive oxygen species)
3. EDCF (Endothelium Derived Constricting Factor)
4. Ang II (Angiotensin II, a vasoconstrictor)

*In diabetes the endothelium is damaged so it produces more harmful species than protective

Question 17

What is a healthy endothelial phenotype?

Answer 17

Anti-coagulant, Anti-Thrombotic, Fibrinolytic

Question 18

What is a dysfunctional endothelial phenotype?

Answer 18

Impaired vasorelaxation, pro-thrombotic, pro-coagulant, pro-inflammatory, insulin resistant

Question 19

What factors influence the development of endothelial dysfunction?

Answer 19

1. Insulin resistance
2. Hypertension
3. Oxidative stress
4. Age
5. Lipids
6. Glucose
7. Inflammatory cytokines

*It has been shown that these factors may also affect the vascular smooth muscle that sits below the endothelium

Question 20

What are some important Diabetes studies to be aware of?

Answer 20

1. UKPDS- the United Kingdom Prospective Diabetes Study
2. DCCT-the Diabetes Control and Complications Trial
3. ACCORD- Action to Control Cardiovascular Risk in Diabetes
4. ADVANCE- Action in Diabetes and Vascular Disease
5. VADT- Veterans Affairs Diabetes Trial

Question 21

What do the UKPDS and DCCT studies show in respect to maintaining levels of glucose control?

Answer 21

These studies both show that maintaining a HbA1c level <7% in diabetes is important for reducing microvascular complications but there is no evidence for benefit to macrovascular complications

Question 22

What do ACCORD, ADVANCE and VADT say in respect to glucose control?

Answer 22

These studies show that intensive glycaemic control had little if any benefit in reducing risk of cardiovascular complications

Question 23

When should glycaemic control be initiated?

Answer 23

EARLY glycaemic control in pre-diabetes seems to be the only way of reducing the macrovascular complications seen in diabetes

Question 24

What is a metabolic memory?

Answer 24

It would seem that endothelial cells are retaining a previous memory of insult by high levels of glucose, EVEN WHEN GLUCOSE IS LOW AND TIGHTLY CONTROLLED
But
evidence is suggesting that this evidence may work the other way too.
Endothelial cells may remember a time of tight glucose control and respond to that even if glucose control is relaxed

Question 25

How did the UKPDS study investigate metabolic memory?

Answer 25

They followed two groups of patients:

1. Tight glycaemic control
2. Conventional glycaemic control

Even when HbA1c levels returned to similar levels after 1 year of the trial ending, the tightly controlled group maintained lower levels of microvascular complications
*This finding indicates that the benefits of tight glycaemic control extend beyond a finite period of intensive managment
UKPDS named this the LEGACY EFFECT
*The DCCT study demonstrated a similar finding but termed it the Metabolic Memory

Question 26

What evidence is there of Metabolic Memory in Cell Culture Models?

Answer 26

(Madi, Cell Physiology, 2009)

• Smooth muscle cells were taken from Saphenous Vein grafts and grown in vitro
• Under light microscopy, the healthy smooth muscle cells can be seen to be SPINDLE SHAPED, but the diabetic smooth muscle cells appeared RHOMBOID SHAPED WITH A MUCH BIGGER SURFACE AREA
• The diabetic cells had much bigger surface areas but there were fewer cells in total

Question 27

What causes the change in Smooth muscle appearance between healthy and diabetes?

Answer 27

These changes have been linked to altered f-Actin
This is a protein involved in the formation of the Cell cytoskeleton
-The healthy SMCs show elongated f-actin
-the Diabetic SMCs show fragmented f-actin
This means that diabetic smooth muscle cells have damaged cytoskeletons

Question 28

What do these changes to smooth muscle cell appearance mean overall?

Answer 28

They mean that the diabetic smooth muscle cells have a poor capacity to replicate which is likely due to their damaged cytoskeleton (f-actin)

Question 29

How is the abnormal SMC profile altered when glucose is tightly controlled?

Answer 29

When glucose is tightly controlled, the diabetic SMCs continue to maintain their altered f-actin phenotype
(although this has only been studied for 3-4 months)

Question 30

What does this lack of altered phenotype mean?

Answer 30

The fact that the diabetic smooth muscle cells don’t change their phenotype even when glucose is tightly controlled indicates a LASTING EFFECT of hyperglycaemia and/or other metabolic disturbances and so provides cell culture evidence that a metabolic memory does exist

Question 31

What are epigenetics?

Answer 31

They are heritable changes in gene expression that do not involve changes to the underlying DNA sequence i.e they are modifications to gene expression rather than changes to the genetic code itself
Epigenetics encompass a change in phenotype without a change in genotype

Question 32

What factors can affect epigenetics?

Answer 32

Age, environment, lifestyle and disease state

Question 33

What is histone modification?

Answer 33

The addition of varying covalent bonds to histone proteins that can impact gene expression by altering chromatin structure

Question 34

What are some epigenetic mechanisms?

Answer 34

MicroRNA expression, Histone Modification, DNA Methylation

Question 35

What are micro RNAs?

Answer 35

They are short, single-stranded non-coding sections of RNA (normally 19-25 nucleotides long)
They are regulators of gene expression but only REDUCE EXPRESSION, they do not increase expression
miRs are species, cell type and tissue specific

Question 36

How do Micro RNAs work?

Answer 36

They cause mRNA degradation or translational repression

Question 37

How are miRs implicated in diabetes?

Answer 37

Research is suggesting that expression of miRs is modulated in a wide range of disease states, including diabetes, and that miRs could be new therapeutic targets

Question 38

What does Riches, JMCC,2014 say about diabetes and miRs?

Answer 38

miR 143 and 145 are found to be significantly more expressed in T2DM smooth muscle cells compared with healthy ones
using anti-miRs 143 & 145, the T2DM SMCs shrink back to spindle type cells
But this is only in vitro
Provides potential for a therapeutic use
Shows that the overexpression of miR 143 and 145 plays a role in the production of altered SMCs in T2DM

Question 39

How are miRs implicated in the limited proliferation of T2DM smcs?

Answer 39

if anti-miR 145 is used, the T2DM cells significantly increase their proliferation rates.
This shows that over expression of miR 145 plays a role in the reduction of proliferation seen in T2DM smcs

Question 40

What causes the miR upregulation in diabetes?

Answer 40

It is likely that the glucose toxicity, insulin, age, lipids and inflammatory mediators all play a role in the misregulation of miRs-because of altered cell signalling pathways and damage to cells resulting in altered dna expression

Question 41

What does this all mean for diabetes and epigenetics?

Answer 41

It means that epigenetic changes can influence candidate genes linked to vascular pathology and thus play a role in metabolic memory and diabetic complications
It presents an explanation for the metabolic memory. An insult changes the expression of a gene, and even if the insult is removed, the expression has already changed so the expression remains